Enzyme treatment of foodstuffs for celiac sprue

ABSTRACT

Administering an effective dose of glutenase to a Celiac or dermatitis herpetiformis patient reduces levels of toxic gluten oligopeptides, thereby attenuating or eliminating the damaging effects of gluten.

GOVERNMENT SUPPORT

This invention was made with Government support under contract 9910949awarded by the National Science Foundation. The Government has certainright in this invention.

BACKGROUND OF THE INVENTION

In 1953, it was first recognized that ingestion of gluten, a commondietary protein present in wheat, barley and rye causes disease insensitive individuals. Gluten is a complex mixture of glutamine- andproline-rich glutenin and prolamine molecules, which is thought to beresponsible for disease induction. Ingestion of such proteins bysensitive individuals produces flattening of the normally luxurious,rug-like, epithelial lining of the small intestine known to beresponsible for efficient and extensive terminal digestion of peptidesand other nutrients. Clinical symptoms of Celiac Sprue include fatigue,chronic diarrhea, malabsorption of nutrients, weight loss, abdominaldistension, anemia, as well as a substantially enhanced risk for thedevelopment of osteoporosis and intestinal malignancies (lymphoma andcarcinoma). The disease has an incidence of approximately 1 in 200 inEuropean populations.

A related disease is dermatitis herpetiformis, which is a chroniceruption characterized by clusters of intensely pruritic vesicles,papules, and urticaria-like lesions. IgA deposits occur in almost allnormal-appearing and perilesional skin. Asymptomatic gluten-sensitiveenteropathy is found in 75 to 90% of patients and in some of theirrelatives. Onset is usually gradual. Itching and burning are severe, andscratching often obscures the primary lesions with eczematization ofnearby skin, leading to an erroneous diagnosis of eczema. Strictadherence to a gluten-free diet for prolonged periods may control thedisease in some patients, obviating or reducing the requirement for drugtherapy. Dapsone, sulfapyridine and colchicines are sometimes prescribedfor relief of itching.

Celiac Sprue is generally considered to be an autoimmune disease and theantibodies found in the serum of the patients supports a theory of animmunological nature of the disease. Antibodies to tissuetransglutaminase (tTG) and gliadin appear in almost 100% of the patientswith active Celiac Sprue, and the presence of such antibodies,particularly of the IgA class, has been used in diagnosis of thedisease.

The large majority of patients express the HLA-DQ2 [DQ(a1*0501, b1*02)]and/or DQ8 [DQ(a1*0301, b1*0302)] molecules. It is believed thatintestinal damage is caused by interactions between specific gliadinoligopeptides and the HLA-DQ2 or DQ8 antigen, which in turn induceproliferation of T lymphocytes in the sub-epithelial layers. T helper 1cells and cytokines apparently play a major role in a local inflammatoryprocess leading to villus atrophy of the small intestine.

At the present time there is no good therapy for the disease, except tocompletely avoid all foods containing gluten. Although gluten withdrawalhas transformed the prognosis for children and substantially improved itfor adults, some people still die of the disease, mainly adults who hadsevere disease at the outset. An important cause of death islymphoreticular disease (especially intestinal lymphoma). It is notknown whether a gluten-free diet diminishes this risk. Apparent clinicalremission is often associated with histologic relapse that is detectedonly by review biopsies or by increased EMA titers.

Gluten is so widely used, for example in commercial soups, sauces, icecreams, hot dogs, and other foods, that patients need detailed lists offoodstuffs to avoid and expert advice from a dietitian familiar withceliac disease. Ingesting even small amounts of gluten may preventremission or induce relapse. Supplementary vitamins, minerals, andhematinics may also be required, depending on deficiency. A few patientsrespond poorly or not at all to gluten withdrawal, either because thediagnosis is incorrect or because the disease is refractory. In thelatter case, oral corticosteroids (e.g., prednisone 10 to 20 mg bid) mayinduce response.

In view of the serious and widespread nature of Celiac Sprue, improvedmethods of treating or ameliorating the effects of the disease areneeded. The present invention addresses such needs.

SUMMARY OF THE INVENTION

The present invention provides methods for treating the symptoms ofCeliac Sprue and/or dermatitis herpetiformis by decreasing the levels oftoxic gluten oligopeptides in foodstuffs, either prior to or afteringestion by a patient. The present invention relates to the discoverythat certain gluten oligopeptides resistant to cleavage by gastric andpancreatic enzymes, that the presence of such peptides results in toxiceffects, and that enzymatic treatment can remove such peptides and theirtoxic effects. By digestion with glutenases, these toxic oligopeptidesare cleaved into fragments, thereby preventing or relieving their toxiceffects in Celiac Sprue or dermatitis herpetiformis patients. In oneaspect of the invention, a foodstuff is treated with a glutenase priorto consumption by the patient. In another aspect of the invention, aglutenase is administered to a patient and acts internally to destroythe toxic oligopeptides. In another aspect of the invention, arecombinant organism that produces a glutenase is administered to apatient. In another aspect of the invention, gene therapy is used toprovide the patient with a gene that expresses a glutenase that destroysthe toxic oligopeptides.

In one aspect, the invention provides methods for the administration ofenteric formulations of one or more glutenases, each of which may bepresent as a single agent or a combination of active agents. In anotheraspect of the invention, stabilized forms of glutenases are administeredto the patient, which stabilized forms are resistant to digestion in thestomach, e.g. to acidic conditions. Alternative methods ofadministration include genetic modification of patient cells, e.g.enterocytes, to express increased levels of peptidases capable ofcleaving immunogenic oligopeptides of gliadin; pretreatment of foodswith glutenases; the introduction of micro-organisms expressing suchpeptidases so as to transiently or permanently colonize the patientintestinal tract; and the like.

In another aspect, the invention provides pharmaceutical formulationscontaining one or more glutenases and a pharmaceutically acceptablecarrier. Such formulations include formulations in which the glutenaseis contained within an enteric coating that allows delivery of theactive agent to the intestine and formulations in which the activeagents are stabilized to resist digestion in acidic stomach conditions.The formulation may comprise one or more glutenases or a mixture or“cocktail” of agents having different activities.

In another aspect, the invention provides foodstuffs derived fromgluten-containing foods that have been treated to remove or to reduce tonon-toxic levels the gluten-derived oligopeptides that are toxic toCeliac Sprue patients, and methods for treating foods to hydrolyze toxicgluten oligopeptides. In other aspects, the invention providesrecombinant microorganisms useful in hydrolyzing the gluten-derivedoligopeptides that are toxic to Celiac Sprue patients from foodstuffs;methods for producing glutenases that digest the gluten-derivedoligopeptides that are toxic to Celiac Sprue patents; purifiedpreparations of the glutenases that digest the gluten-derivedoligopeptides that are toxic to Celiac Sprue patents; and recombinantvectors that code for the expression of glutenases that digest thegluten-derived oligopeptides that are toxic to Celiac Sprue patents.

These and other aspects and embodiments of the invention are describedin more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Brush border membrane catalyzed digestion of theimmunodominant gliadin peptide. FIG. 1A: LC-MS traces of peptides asshown, after digestion with 27 ng/μl rat brush border membrane (BBM)protein for the indicated time. Reaction products were separated byreversed phase HPLC and detected by mass spectroscopy (ion countsm/z=300-2000 g/mol). The indicated peptide fragments were confirmed bycharacteristic tandem MS fragmentation patterns. The SEQ ID NO:2pyroQLQPFPQPQLPY peak corresponds to an N-terminally pyroglutaminatedspecies, which is generated during HPLC purification of the syntheticstarting material. FIG. 1B: Abundance of individual digestion productsas a function of time. The peptide fragments in FIG. 1A were quantifiedby integrating the corresponding MS peak area (m/z=300-2000 g/mol). Theresulting MS intensities are plotted as a function of digestion time(with BBM only). The digestion experiment was repeated in the presenceof exogenous DPP IV from Aspergillus fumigatus (Chemicon International,CA, 0.28 μU DPP IV/ng BBM protein) and analyzed as above (open bars).The relative abundance of different intermediates could be estimatedfrom the UV₂₈₀ traces and control experiments using authentic standards.The inserted scheme shows an interpretative diagram of the digestionpathways of SEQ ID NO:1) QLQPFPQPQLPY and its intermediates, the BBMpeptidases involved in each step, and the amino acid residues that arereleased. The preferred breakdown pathway is indicated in bold.APN=aminopeptidase N, CPP=carboxypeptidase P, DPP IV=dipeptidyldipeptidase IV.

FIG. 2A-2B. C-terminal digestion of the immunodominant gliadin peptideby brush border membrane. FIG. 2A: (SEQ ID NO:3) PQPQLPYPQPQLPY wasdigested by 27 ng/μl brush border membrane (BBM) protein preparationsfor the indicated time and analyzed as in FIG. 1A. The identity of thestarting material and the product (SEQ ID NO:4) PQPQLPYPQPQLP wascorroborated by MSMS fragmentation. The intrinsic mass intensities ofthe two peptides were identical, and the UV₂₈₀ extinction coefficient of(SEQ ID NO:4) PQPQLPYPQPQLP was half of the starting material inaccordance with the loss of one tyrosine. All other intermediates were≦1%. The scheme below shows the proposed BBM digestion pathway of (SEQID NO:3) PQPQLPYPQPQLPY with no observed N-terminal processing (crossedarrow) and the removal of the C-terminal tyrosine by carboxypeptidaseP(CPP) in bold. Further C-terminal processing by dipeptidylcarboxypeptidase (DCP) was too slow to permit analysis of the subsequentdigestion steps (dotted arrows). FIG. 2B: Influence of dipeptidylcarboxypeptidase on C-terminal digestion. (SEQ ID NO:3) PQPQLPYPQPQLPYin phosphate buffered saline:Tris buffered saline=9:1 was digested byBBM alone or with addition of exogenous rabbit lung DCP (CortexBiochemicals, CA) or captopril. After overnight incubation, the fractionof accumulated SEQ ID NO:4) PQPQLPYPQPQLP (compared to initial amountsof (SEQ ID NO:3) PQPQLPYPQPQLPY at t=0 min) was analyzed as in FIG. 2A,but with an acetonitrile gradient of 20-65% in 6-35 minutes.

FIG. 3. Dose dependent acceleration of brush border mediated digestionby exogenous endoproteases. As seen from FIG. 2A-2B, the peptide (SEQ IDNO:4) PQPQLPYPQPQLP is stable toward further digestion. This peptide wasdigested with 27 ng/μl brush border membranes, either alone, withincreasing amounts of exogenous prolyl endopeptidase (PEP, specificactivity 28 μU/pg) from Flavobacterium meningosepticum (US Biological,MA), or with additional elastase (E-1250, Sigma, Mo.), bromelain(B-5144, Sigma, Mo.) or papain (P-5306, Sigma, Mo.) (12). After onehour, the fraction of remaining (SEQ ID NO:4) PQPQLPYPQPQLP (compared tothe initial amount at t=0 min) was analyzed and quantified as in FIG. 1.

FIG. 4. Products of gastric and pancreatic protease mediated digestionof 2-gliadin under physiological conditions. Analysis was performed byLC-MS. The longest peptides are highlighted by arrows and also in thesequence of 2-gliadin (inset).

FIG. 5. In vivo brush border membrane digestion of peptides. LC-UV215traces of 25 M of (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPFbefore perfusion and after perfusion (residence time=20 min). LC-UV215traces of 50 M of SEQ ID NO:1 QLQPFPQPQLPY before perfusion and afterperfusion (residence time=20 min).

FIG. 6. Alignment of representative gluten and non-gluten peptideshomologous to (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.

FIG. 7. Breakdown and detoxification of 33-mer gliadin peptide with PEP.In vitro incubation of PEP (540 mU/ml) with the 33-mer gliadin peptide(100 M) for the indicated time. In vivo digestion of the 33-mer gliadinpeptide (25 M) with PEP (25 mU/ml) and the rat's intestine (residencetime=20 min).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Celiac Sprue and/or dermatitis herpetiformis are treated by digestion ofgluten oligopeptides contained in foodstuffs consumed by individualssuffering from one or both conditions. Gluten oligopeptides are highlyresistant to cleavage by gastric and pancreatic peptidases such aspepsin, trypsin, chymotrypsin, and the like. By providing for digestionof gluten oligopeptides with glutenase, oligopeptides are cleaved intofragments, thereby preventing the disease-causing toxicity.

Methods and compositions are provided for the administration of one ormore glutenases inhibitors to a patient suffering from Celiac Sprueand/or dermatitis herpetiformis. In some patients, these methods andcompositions will allow the patient to ingest glutens without serioushealth consequences, much the same as individuals that do not sufferfrom either of these conditions. In some embodiments, the formulationsof the invention comprise a glutenase contained in an enteric coatingthat allows delivery of the active agent(s) to the intestine; in otherembodiments, the active agent(s) is stabilized to resist digestion inacidic stomach conditions. In some cases the active agent(s) havehydrolytic activity under acidic pH conditions, and can thereforeinitiate the proteolytic process on toxic gluten sequences in thestomach itself. Alternative methods of administration provided by theinvention include genetic modification of patient cells, e.g.enterocytes, to express increased levels of glutenases; and theintroduction of micro-organisms expressing such glutenases so as totransiently or permanently colonize the patient's intestinal tract. Suchmodified patient cells (which include cells that are not derived fromthe patient but that are not immunologically rejected when administeredto the patient) and microorganisms of the invention are, in someembodiments, formulated in a pharmaceutically acceptable excipient, orintroduced in foods. In another embodiment, the invention provides foodspretreated or combined with a glutenase and methods for treating foodsto remove the toxic oligopeptides of gluten.

The methods of the invention can be used for prophylactic as well astherapeutic purposes. As used herein, the term “treating” refers both tothe prevention of disease and the treatment of a disease or apre-existing condition. The invention provides a significant advance inthe treatment of ongoing disease, to stabilize or improve the clinicalsymptoms of the patient. Such treatment is desirably performed prior toloss of function in the affected tissues but can also help to restorelost function or prevent further loss of function. Evidence oftherapeutic effect may be any diminution in the severity of disease,particularly as measured by the severity of symptoms such as fatigue,chronic diarrhea, malabsorption of nutrients, weight loss, abdominaldistension, anemia, and other symptoms of Celiac Sprue. Other diseaseindicia include the presence of antibodies specific for glutens, thepresence of antibodies specific for tissue transglutaminase, thepresence of pro-inflammatory T cells and cytokines, damage to the villusstructure of the small intestine as evidenced by histological or otherexamination, enhanced intestinal permeability, and the like.

Patients that can benefit from the present invention may be of any ageand include adults and children. Children in particular benefit fromprophylactic treatment, as prevention of early exposure to toxic glutenpeptides can prevent initial development of the disease. Childrensuitable for prophylaxis can be identified by genetic testing forpredisposition, e.g. by HLA typing; by family history, by T cell assay,or by other medical means. As is known in the art, dosages may beadjusted for pediatric use.

Although the present invention is not to be bound by any theory ofaction, it is believed that the primary event in Celiac Sprue requirescertain gluten oligopeptides to access antigen binding sites within thelamina propria region interior to the relatively impermeable surfaceintestinal epithelial layer. Ordinarily, oligopeptide end products ofpancreatic protease processing are rapidly and efficiently hydrolyzedinto amino acids and/or di- or tri-peptides by gastric peptidases beforethey are transported across the epithelial layer. However, glutens areparticularly peptidase resistant, which may be attributed to the usuallyhigh proline content of these proteins, a residue that is inaccessibleto most gastric peptidases.

The normal assimilation of dietary proteins by the human gut can bedivided into three major phases: (i) initiation of proteolysis in thestomach by pepsin and highly efficient endo- and C-terminal cleavage inthe upper small intestine cavity (duodenum) by secreted pancreaticproteases and carboxypeptidases; (ii) further processing of theresulting oligopeptide fragments by exo- and endopeptidases anchored inthe brush border surface membrane of the upper small intestinalepithelium (jejunum); and (iii) facilitated transport of the resultingamino acids, di- and tripeptides across the epithelial cells into thelamina propria, from where these nutrients enter capillaries fordistribution throughout the body. Because most proteases and peptidasesnormally present in the human stomach and small intestine are unable tohydrolyze the amide bonds of proline residues, it is shown herein thatthe abundance of proline residues in gliadins and related proteins fromwheat, rye and barley can constitute a major digestive obstacle for theenzymes involved in phases (i) and (ii) above. This leads to anincreased concentration of relatively stable gluten derivedoligopeptides in the gut. Furthermore, because aminopeptidase andespecially carboxypeptidase activity towards oligopeptides with prolineresidues at the N- and C-termini, respectively, is low in the smallintestine, detoxification of gluten oligopeptides in phase (iii) aboveis also slow. By administering peptidases capable of cleaving suchgluten oligopeptides in accordance with the methods of the invention,the amount of toxic peptides is diminished, thereby slowing or blockingdisease progression.

Tissue transglutaminase (tTGase), an enzyme found on the extracellularsurface in many organs including the intestine, catalyzes the formationof isopeptide bonds between glutamine and lysine residues of differentpolypeptides, leading to protein-protein crosslinks in the extracellularmatrix. The enzyme tTGase is the primary focus of the autoantibodyresponse in Celiac Sprue. Gliadins, secalins and hordeins containseveral sequences rich in Pro-Gln residues that are high-affinitysubstrates for tTGase; tTGase catalyzed deamidation of at least some ofthese sequences dramatically increases their affinity for HLA-DQ2, theclass II MHC allele present in >90% Celiac Sprue patients. Presentationof these deamidated epitopes by DQ2 positive antigen presenting cellseffectively stimulates proliferation of gliadin-specific T cells fromintestinal biopsies of most Celiac Sprue patients. The toxic effects ofgluten include immunogenicity of the gluten oligopeptides, leading toinflammation; the lectin theory predicts that gliadin peptides may alsodirectly bind to surface receptors.

The present invention relates generally to methods and reagents usefulin treating foodstuffs containing gluten with enzymes that digest theoligopeptides toxic to Celiac Sprue patients. Although specific enzymesare exemplified herein, any of a number of alternative enzymes andmethods apparent to those of skill in the art upon contemplation of thisdisclosure are equally applicable and suitable for use in practicing theinvention. The methods of the invention, as well as tests to determinetheir efficacy in a particular patient or application, can be carriedout in accordance with the teachings herein using procedures standard inthe art. Thus, the practice of the present invention may employconventional techniques of molecular biology (including recombinanttechniques), microbiology, cell biology, biochemistry and immunologywithin the scope of those of skill in the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Weir & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction” (Mullis et al., eds., 1994); and “Current Protocols inImmunology” (J. E. Coligan et al., eds., 1991); as well as updated orrevised editions of all of the foregoing.

As used herein, the term “glutenase” refers to an enzyme useful in themethods of the present invention that is capable, alone or incombination with endogenous or exogenously added enzymes, of cleavingtoxic oligopeptides of gluten proteins of wheat, barley, oats and ryeinto non-toxic fragments. Gluten is the protein fraction in cerealdough, which can be subdivided into glutenins and prolamines, which aresubclassified as gliadins, secalins, hordeins, and avenins from wheat,rye, barley and oat, respectively. For further discussion of glutenproteins, see the review by Wieser (1996) Acta Paediatr Suppl. 412:3-9,incorporated herein by reference.

In one embodiment, the term “glutenase” as used herein refers to aprotease or a peptidase enzyme that meets one or more of the criteriaprovided herein. Using these criteria, one of skill in the art candetermine the suitability of a candidate enzyme for use in the methodsof the invention. Many enzymes will meet multiple criteria, includingtwo, three, four or more of the criteria, and some enzymes will meet allof the criteria. The terms “protease” or “peptidase” can refer to aglutenase and as used herein describe a protein or fragment thereof withthe capability of cleaving peptide bonds, where the scissile peptidebond may either be terminal or internal in oligopeptides or largerproteins. Prolyl-specific peptidases are glutenases useful in thepractice of the present invention.

Glutenases of the invention include protease and peptidase enzymeshaving at least about 20% sequence identity at the amino acid level,more usually at least about 40% sequence identity, and preferably atleast about 70% sequence identity to one of the following peptidases:prolyl endopeptidase (PEP) from F. meningosepticum (Genbank accessionnumber D10980), PEP from A. hydrophila (Genbank accession numberD14005), PEP form S. caplsulata (Genbank accession number AB010298), DCPI from rabbit (Genbank accession number X62551), DPP IV from Aspergillusfumigatus (Genbank accession number U87950) or cysteine proteinase Bfrom Hordeum vulgare (Genbank accession number JQ1110).

In one embodiment of the present invention, the glutenase is a PEP.Homology-based identification (for example, by a PILEUP sequenceanalysis) of prolyl endopeptidases can be routinely performed by thoseof skill in the art upon contemplation of this disclosure to identifyPEPs suitable for use in the methods of the present invention. PEPs areproduced in microorganisms, plants and animals. PEPs belong to theserine protease superfamily of enzymes and have a conserved catalytictriad composed of a Ser, His, and Asp residues. Some of these homologshave been characterized, e.g. the enzymes from F. meningoscepticum,Aeromonas hydrophila, Aeromonas punctata, Novosphingobium capsulatum,Pyrococcus furiosus and from mammalian sources are biochemicallycharacterized PEPs. Others such as the Nostoc and Arabidopsis enzymesare likely to be PEPs but have not been fully characterized to date. Yetothers, such as the E. coli and M. xanthus enzymes, may not be PEPs butare homologous members of the serine protease superfamily, and can beuseful starting materials in protein engineering to make a PEP useful inthe practice of the present invention. Relative to the F.meningoscepticum enzyme, the pairwise sequence identity of this familyof enzymes is in the 30-60% range. Accordingly, PEPs include enzymeshaving >30% identity to the F. meningoscepticum enzyme (as in thePyrococcus enzymes), or having >40% identity (as in the Novosphingobiumenzymes), or having >50% identity (as in the Aeromonas enzymes) to theF. meningoscepticum enzyme.

A glutenase of the invention includes a peptidase or protease that has aspecific activity of at least 2.5 U/mg, preferably 25 U/mg and morepreferably 250 U/mg for cleavage of a peptide comprising one of more ofthe following motifs: Gly-Pro-pNA, Z-Gly-Pro-pNA (where Z is abenzyloxycarbonyl group), and Hip-His-Leu, where “Hip” is hippuric acid,pNA is para-nitroanilide, and 1 U is the amount of enzyme required tocatalyze the turnover of 1 mole of substrate per minute.

A glutenase of the invention includes an enzyme belonging to any of thefollowing enzyme classifications: EC 3.4.21.26, EC 3.4.14.5, or EC3.4.15.1.

A glutenase of the invention includes an enzyme having a kcat/Km of atleast about 2.5 s−1 M−1, usually at least about 250 s−1 M−1 andpreferably at least about 25000 s−1 M−1 for cleavage of any of thefollowing peptides under optimal conditions: (SEQ ID NO:1) QLQPFPQPQLPY,(SEQ ID NO:3) PQPQLPYPQPQLPY, (SEQ ID NO:13) QPQQSFPQQQ, (SEQ ID NO:14)QLQPFPQPELPY, (SEQ ID NO:15) PQPELPYPQPELPY, (SEQ ID NO:16) QPQQSFPEQQ.A glutenase of the invention includes peptidase or protease having aspecificity kcat/Km>2 mM⁻¹s⁻¹ for the quenched fluorogenic substrateAbz-QPQQP-Tyr(NO₂)-D.

A glutenase useful in the practice of the present invention can beidentified by its ability to cleave a pretreated substrate to removetoxic gluten oligopeptides, where a “pretreated substrate” is a gliadin,hordein, secalin or avenin protein that has been treated withphysiological quantities of gastric and pancreatic proteases, includingpepsin (1:100 mass ratio), trypsin (1:100), chymotrypsin (1:100),elastase (1:500), and carboxypeptidases A and B (1:100). Pepsindigestion may be performed at pH 2 for 20 min., to mimic gastricdigestion, followed by further treatment of the reaction mixture withtrypsin, chymotrypsin, elastase and carboxypeptidase at pH 7 for 1 hour,to mimic duodenal digestion by secreted pancreatic enzymes. Thepretreated substrate comprises oligopeptides resistant to digestion,e.g. under physiological conditions.

The ability of a peptidase or protease to cleave a pretreated substratecan be determined by measuring the ability of an enzyme to increase theconcentration of free NH₂-termini in a reaction mixture containing 1mg/ml pretreated substrate and 10 g/ml of the peptidase or protease,incubated at 37° C. for 1 hour. A glutenase useful in the practice ofthe present invention will increase the concentration of the free aminotermini under such conditions, usually by at least about 25%, moreusually by at least about 50%, and preferably by at least about 100%. Aglutenase includes an enzyme capable of reducing the residual molarconcentration of oligopeptides greater than about 1000 Da in a 1 mg/ml“pretreated substrate” after a 1 hour incubation with 10 μg/ml of theenzyme by at least about 2-fold, usually by at least about 5-fold, andpreferably by at least about 10-fold. The concentration of sucholigopeptides can be estimated by methods known in the art, for examplesize exclusion chromatography and the like.

A glutenase of the invention includes an enzyme capable of reducing thepotency by which a “pretreated substrate” can antagonize binding of (SEQID NO:17) PQPELPYPQPQLP to HLA-DQ2. The ability of a substrate to bindto HLA-DQ is indicative of its toxicity; fragments smaller than about 8amino acids are generally not stably bound to Class II MHC. Treatmentwith a glutenase that digests toxic oligopeptides, by reducing theconcentration of the toxic oligopeptides, prevents a mixture containingthem from competing with a test peptide for MHC binding. To test whethera candidate glutenase can be used for purposes of the present invention,a 1 mg/ml solution of “pretreated substrate” may be first incubated with10 μg/ml of the candidate glutenase, and the ability of the resultingsolution to displace radioactive (SEQ ID NO:18) PQPELPYPQPQPLP pre-boundto HLA-DQ2 molecules can then be quantified, with a reduction ofdisplacement, relative to a non-treated control, indicative of utilityin the methods of the present invention.

A glutenase of the invention includes an enzyme that reduces theanti-tTG antibody response to a “gluten challenge diet” in a CeliacSprue patient by at least about 2-fold, more usually by at least about5-fold, and preferably by at least about 10-fold. A “gluten challengediet” is defined as the intake of 100 g bread per day for 3 days by anadult Celiac Sprue patient previously on a gluten-free diet. Theanti-tTG antibody response can be measured in peripheral blood usingstandard clinical diagnostic procedures, as known in the art.

Excluded from the term “glutenase” are the following peptidases: humanpepsin, human trypsin, human chymotrypsin, human elastase, papayapapain, and pineapple bromelain, and usually excluded are enzymes havinggreater than 98% sequence identity at the amino acid level to suchpeptidases, more usually excluded are enzymes having greater than 90%sequence identity at the amino acid level to such peptidases, andpreferably excluded are enzymes having greater than 70% sequenceidentity at the amino acid level to such peptidases.

Among gluten proteins with potential harmful effect to Celiac Spruepatients are included the storage proteins of wheat, species of whichinclude Triticum aestivum; Triticum aethiopicum; Triticum baeoticum;Triticum militinae; Triticum monococcum; Triticum sinskajae; Triticumtimopheevii; Triticum turgidum; Triticum urartu, Triticum vavilovii;Triticum zhukovskyi; etc. A review of the genes encoding wheat storageproteins may be found in Colot (1990) Genet Eng (NY) 12:225-41. Gliadinis the alcohol-soluble protein fraction of wheat gluten. Gliadins aretypically rich in glutamine and proline, particularly in the N-terminalpart. For example, the first 100 amino acids of α- and γ-gliadinscontain ˜35% and ˜20% of glutamine and proline residues, respectively.Many wheat gliadins have been characterized, and as there are manystrains of wheat and other cereals, it is anticipated that many moresequences will be identified using routine methods of molecular biology.In one aspect of the present invention, genetically modified plants areprovided that differ from their naturally occurring counterparts byhaving gliadin proteins that contain a reduced content of glutamine andproline residues.

Examples of gliadin sequences include but are not limited to wheat alphagliadin sequences, for example as provided in Genbank, accession numbersAJ133612; AJ 133611; AJ133610; AJ133609; AJ 133608; AJ133607; AJ133606;AJ133605; AJ133604; AJ 133603; AJ133602; D84341.1; U51307; U51306;U51304; U51303; U50984; and U08287. A sequence of wheat omega gliadin isset forth in Genbank accession number AF280605.

For the purposes of the present invention, toxic gliadin oligopeptidesare peptides derived during normal human digestion of gliadins andrelated storage proteins as described above, from dietary cereals, e.g.wheat, rye, barley, and the like. Such oligopeptides are believed to actas antigens for T cells in Celiac Sprue. For binding to Class II MHCproteins, immunogenic peptides are usually from about 8 to 20 aminoacids in length, more usually from about 10 to 18 amino acids. Suchpeptides may include PXP motifs, such as the motif PQPQLP (SEQ ID NO:8).Determination of whether an oligopeptide is immunogenic for a particularpatient is readily determined by standard T cell activation and otherassays known to those of skill in the art.

As demonstrated herein, during digestion, peptidase resistantoligopeptides remain after exposure of glutens, e.g. gliadin, to normaldigestive enzymes. Examples of peptidase resistant oligopeptides areprovided, for example, as set forth in SEQ ID NO:5, 6, 7 and 10. Otherexamples of immunogenic gliadin oligopeptides are described in Wieser(1995) Baillieres Clin Gastroenterol 9(2):191-207, incorporated hereinby reference.

Determination of whether a candidate enzyme will digest a toxic glutenoligopeptide, as discussed above, can be empirically determined. Forexample, a candidate may be combined with an oligopeptide comprising oneor more Gly-Pro-pNA, Z-Gly-Pro-pNA, Hip-His-Leu, Abz-QLP-Tyr(NO₂)-PQ,Abz-PYPQPQ-Tyr(NO₂), PQP-Lys(Abz)-LP-Tyr(NO₂)-PQPQLP,PQPQLP-Tyr(NO₂)-PQP-Lys(Abz)-LP motifs; with one or more of theoligopeptides (SEQ ID NO:1) QLQPFPQPQLPY, (SEQ ID NO:3) PQPQLPYPQPQLPY,(SEQ ID NO:13) QPQQSFPQQQ, (SEQ ID NO:14) QLQPFPQPELPY, (SEQ ID NO:15)PQPELPYPQPELPY, (SEQ ID NO:16) QPQQSFPEQQ or (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF; or with a pretreated substratecomprising one or more of gliadin, hordein, secalin or avenin proteinsthat have been treated with physiological quantities of gastric andpancreatic proteases. In each instance, the candidate is determined tobe a glutenase of the invention if it is capable of cleaving theoligopeptide. Glutenases that have a low toxicity for human cells andare active in the physiologic conditions present in the intestinal brushborder are preferred for use in some applications of the invention, andtherefore it may be useful to screen for such properties in candidateglutenases.

The oligopeptide or protein substrates for such assays may be preparedin accordance with conventional techniques, such as synthesis,recombinant techniques, isolation from natural sources, or the like. Forexample, solid-phase peptide synthesis involves the successive additionof amino acids to create a linear peptide chain (see Merrifield (1963)J. Am. Chem. Soc. 85:2149-2154). Recombinant DNA technology can also beused to produce the peptide.

Candidate glutenases for use in the practice of the present inventioncan be obtained from a wide variety of sources, including libraries ofnatural and synthetic proteins. For example, numerous means areavailable for random and directed mutation of proteins. Alternatively,libraries of natural proteins in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Extracts ofgerminating wheat and other grasses is of interest as a source ofcandidate enzymes. Natural or synthetically produced libraries andcompounds are readily modified through conventional chemical, physicaland biochemical means, and such means can be used to producecombinatorial libraries. Known pharmacological agents may be subjectedto directed or random chemical modifications, such as acylation,alkylation, esterification, and amidification, to produce structuralanalogs of proteins.

Generally, a variety of assay mixtures are run in parallel withdifferent peptidase concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e. at zero concentration or below thelevel of detection. A variety of other reagents may be included in ascreening assay. These include reagents like salts, detergents, and thelike that are used to facilitate optimal activity and/or reducenon-specific or background interactions. Reagents that improve theefficiency of the assay may be used. The mixture of components is addedin any order that provides for the requisite activity. Incubations areperformed at any suitable temperature, typically between 4 and 40° C.Incubation periods are selected for optimum activity but can also beoptimized to facilitate rapid high-throughput screening or otherpurposes. Typically, between 0.1 and 1 hours will be sufficient.

The level of digestion of the toxic oligopeptide can be compared to abaseline value. The disappearance of the starting material and/or thepresence of digestion products can be monitored by conventional methods.For example, a detectable marker can be conjugated to a peptide, and thechange in molecular weight associated with the marker is thendetermined, e.g. acid precipitation, molecular weight exclusion, and thelike. The baseline value can be a value for a control sample or astatistical value that is representative a control population. Variouscontrols can be conducted to ensure that an observed activity isauthentic, including running parallel reactions, positive and negativecontrols, dose response, and the like.

Active glutenases identified by the screening methods described hereincan serve as lead compounds for the synthesis of analog compounds toidentify glutenases with improved properties. Identification of analogcompounds can be performed through use of techniques such asself-consistent field (SCF) analysis, configuration interaction (CI)analysis, and normal mode dynamics analysis.

In one embodiment of the invention, the glutenase is a prolylendopeptidase (PEP, EC 3.4.21.26). Prolyl endopeptidases are widelydistributed in microorganisms, plants and animals, and have been clonedfrom Flavobacterium meningosepticum, (Yoshimoto et al. (1991) J.Biochem. 110, 873-8); Aeromonas hydrophyla (Kanatani et al. (1993) J.Biochem. 113, 790-6); Sphingomonas capsulata (Kabashima et al. (1998)Arch. Biochem. Biophys. 358, 141-148), Pyrococcus furious (Robinson etal. (1995) Gene 152, 103-6); pig (Rennex et al. (1991) Biochemistry 30,2195-2030); and the like. The suitability of a particular enzyme isreadily determined by the assays described above, by clinical testing,determination of stability in formulations, and the like. Other sourcesof PEP include Lactobacilli (Habibi-Najafi et al. (1994) J. Dairy Sci.77, 385-392), from where the gene of interest can be readily clonedbased on sequence homology to the above PEP's or via standard reversegenetic procedures involving purification, amino-acid sequencing,reverse translation, and cloning of the gene encoding the targetextracellular enzyme.

In another embodiment of the invention, glutenases are peptidasespresent in the brush border, which are supplemented. Formulations ofinterest may comprise such enzymes in combination with other peptidases.Peptidases present in brush border include dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), and dipeptidyl carboxypeptidase (DCP, EC 3.4.15.1).The human form of these proteins may be used, or modified forms may beisolated from other suitable sources. Example of DPP IV enzymes includeAspergillus spp. (e.g. Byun et al. (2001) J. Agric. Food Chem. 49,2061-2063), ruminant bacteria such as Prevotella albensis M384 (NCBIprotein database Locus #CAC42932), dental bacteria such as Porphyromonasgingivalis W83 (Kumugai et al. (2000) Infect. Immun. 68, 716-724),lactobacilli such as Lactobacillus helveticus (e.g. Vesanto, et al,(1995) Microbiol. 141, 3067-3075), and Lactococcus lactis (Mayo et al.,(1991) Appl. Environ. Microbiol. 57, 38-44). Other DPP IV candidates canreadily be recognized based on homology to the above enzymes,preferably >30% sequence identity. Similarly, secreted dipeptidylcarboxypeptidases that cleave C-terminal X-Pro sequences are found inmany microbial sources including Pseudomonas spp (e.g. Ogasawara et al,(1997) Biosci. Biotechnol. Biochem. 61, 858-863), Streptomyces spp.(e.g. Miyoshi et al., (1992) J. Biochem. 112, 253-257) and Aspergillispp. (e.g. Ichishima et al., (1977) J. Biochem. 81, 1733-1737). Ofparticular interest is the enzyme from Aspergillus saitoi (Ichishima),due to its high activity at acidic pH values. Although the genesencoding many of these enzymes have not yet been cloned, they can bereadily cloned by standard reverse genetic procedures. The DCP I enzymescan be purified from the extracellular medium based on their ability tohydrolyze (SEQ ID NO:19) Z-Gly-Pro-Leu-Gly-Pro, Z-Gly-Pro, orHip-Gly-Pro. Alternatively, putative DCP I genes can be identified basedon homology to the E. coli enzyme (NCBI protein database LocusCAA41014.)

In another embodiment of the invention, glutenases are endoproteasesfound in developing grains of toxic cereals such as wheat, barley andrye. For example, Dominguez and Cejudo (Plant Physiol. 112, 1211-1217,1996) have shown that the endosperm of wheat (i.e. the part of the grainthat contains gliadin and glutenin) contains a variety of neutral andacid proteases. Although these proteases have not been individuallycharacterized, they are expected to be an especially rich source ofglutenases. Moreover, although the genes encoding these proteases havenot yet been cloned, Dominguez and Cejudo have established a convenientSDS-PAGE assay for identification and separation of these proteases.After excision of the corresponding protein bands from the gel, limitedsequence information can be obtained. The cDNA encoding these proteasescan therefore be readily cloned from this information using establishedreverse genetic procedures, and expressed in heterologous bacterial orfungal hosts. Of particular interest are proteases that hydrolyzeα2-gliadin within the 33-mer amino acid sequence identified in Example 2below. Of further interest are the subset of these proteases that retainactivity at acidic pH values (pH2-5) encountered in the stomach.

The amino acid sequence of a glutenase, e.g. a naturally occurringglutenase, can be altered in various ways known in the art to generatetargeted changes in sequence and additional glutenase enzymes useful inthe formulations and compositions of the invention. Such variants willtypically be functionally-preserved variants, which differ, usually insequence, from the corresponding native or parent protein but stillretain the desired biological activity. Variants also include fragmentsof a glutenase that retain enzymatic activity. Various methods known inthe art can be used to generate targeted changes, e.g. phage display incombination with random and targeted mutations, introduction of scanningmutations, and the like.

A variant can be substantially similar to a native sequence, i.e.differing by at least one amino acid, and can differ by at least two butusually not more than about ten amino acids (the number of differencesdepending on the size of the native sequence). The sequence changes maybe substitutions, insertions or deletions. Scanning mutations thatsystematically introduce alanine, or other residues, may be used todetermine key amino acids. Conservative amino acid substitutionstypically include substitutions within the following groups: (glycine,alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid);(asparagine, glutamine); (serine, threonine); (lysine, arginine); and(phenylalanine, tyrosine).

Glutenase fragments of interest include fragments of at least about 20contiguous amino acids, more usually at least about 50 contiguous aminoacids, and may comprise 100 or more amino acids, up to the completeprotein, and may extend further to comprise additional sequences. Ineach case, the key criterion is whether the fragment retains the abilityto digest the toxic oligopeptides that contribute to the symptoms ofCeliac Sprue.

Modifications of interest that do not alter primary sequence includechemical derivatization of proteins, e.g., acetylation or carboxylation.Also included are modifications of glycosylation, e.g. those made bymodifying the glycosylation patterns of a protein during its synthesisand processing or in further processing steps; e.g. by exposing theprotein to enzymes that affect glycosylation, such as mammalianglycosylating or deglycosylating enzymes. Also embraced are sequencesthat have phosphorylated amino acid residues, e.g. phosphotyrosine,phosphoserine, or phosphothreonine.

Also useful in the practice of the present invention are proteins thathave been modified using molecular biological techniques and/orchemistry so as to improve their resistance to proteolytic degradationand/or to acidic conditions such as those found in the stomach, and tooptimize solubility properties or to render them more suitable as atherapeutic agent. For example, the backbone of the peptidase can becyclized to enhance stability (see Friedler et al. (2000) J. Biol. Chem.275:23783-23789). Analogs of such proteins include those containingresidues other than naturally occurring L-amino acids, e.g. D-aminoacids or non-naturally occurring synthetic amino acids.

The glutenase proteins of the present invention may be prepared by invitro synthesis, using conventional methods as known in the art. Variouscommercial synthetic apparatuses are available, for example, automatedsynthesizers by Applied Biosystems, Inc., Foster City, Calif., Beckman,and other manufacturers. Using synthesizers, one can readily substitutefor the naturally occurring amino acids one or more unnatural aminoacids. The particular sequence and the manner of preparation will bedetermined by convenience, economics, purity required, and the like. Ifdesired, various groups can be introduced into the protein duringsynthesis that allow for linking to other molecules or to a surface. Forexample, cysteines can be used to make thioethers, histidines can beused for linking to a metal ion complex, carboxyl groups can be used forforming amides or esters, amino groups can be used for forming amides,and the like.

The glutenase proteins useful in the practice of the present inventionmay also be isolated and purified in accordance with conventionalmethods from recombinant production systems and from natural sources. Alysate can be prepared from the expression host and the lysate purifiedusing HPLC, exclusion chromatography, gel electrophoresis, affinitychromatography, and/or other purification techniques. Typically, thecompositions used in the practice of the invention will comprise atleast 20% by weight of the desired product, more usually at least about75% by weight, preferably at least about 95% by weight, and fortherapeutic purposes, usually at least about 99.5% by weight, inrelation to contaminants related to the method of preparation of theproduct and its purification. Usually, the percentages will be basedupon total protein.

In one aspect, the present invention provides a purified preparation ofa glutenase. Prior to the present invention, there was no need for aglutenase that could be ingested by a human or mixed with a foodstuff.Thus, prior to the present invention most glutenases did not exist in aform free of contaminants that could be deleterious to a human ifingested. The present invention creates a need for such glutenasepreparations and provides them and methods for preparing them. In arelated embodiment, the present invention provides novel foodstuffs thatare derived from gluten-containing foodstuffs but have been treated toreduce the concentration and amount of the oligopeptides andoligopeptide sequences discovered to be toxic to Celiac Sprue patients.While gluten-free or reduced-gluten content foods have been made, thefoodstuffs of the present invention differ from such foodstuffs not onlyby the manner in which they are prepared, by treatment of the foodstuffwith a glutenase, but also by their content, as the methods of the priorart result in alteration of non-toxic (to Celiac Sprue patients)components of the foodstuff, resulting in a different taste andcomposition. Prior art foodstuffs include, for example, CodexAlimentarius wheat starch, which is available in Europe and has <100 ppmgluten. The starch is usually prepared by processes that take advantageof the fact that gluten is insoluble in water whereas starch is soluble.

In one embodiment of the present invention, a Celiac Sprue patient is,in addition to being provided a glutenase or food treated in accordancewith the present methods, provided an inhibitor of tissuetransglutaminase, an anti-inflammatory agent, an anti-ulcer agent, amast cell-stabilizing agents, and/or and an-allergy agent. Examples ofsuch agents include HMG-CoA reductase inhibitors with anti-inflammatoryproperties such as compactin, lovastatin, simvastatin, pravastatin andatorvastatin; anti-allergic histamine H1 receptor antagonists such asacrivastine, cetirizine, desloratadine, ebastine, fexofenadine,levocetirizine, loratadine and mizolastine; leukotriene receptorantagonists such as montelukast and zafirlukast; COX2 inhibitors such ascelecoxib and rofecoxib; p38 MAP kinase inhibitors such as BIRB-796; andmast cell stabilizing agents such as sodium chromoglycate (chromolyn),pemirolast, proxicromil, repirinast, doxantrazole, amlexanox nedocromiland probicromil.

As used herein, compounds which are “commercially available” may beobtained from commercial sources including but not limited to AcrosOrganics (Pittsburgh Pa.), Aldrich Chemical (Milwaukee Wis., includingSigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), AvocadoResearch (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet(Cornwall, U.K.), Chemservice Inc. (West Chester Pa.), Crescent ChemicalCo. (Hauppauge N.Y.), Eastman Organic Chemicals, Eastman Kodak Company(Rochester N.Y.), Fisher Scientific Co. (Pittsburgh Pa.), FisonsChemicals (Leicestershire UK), Frontier Scientific (Logan Utah), ICNBiomedicals, Inc. (Costa Mesa Calif.), Key Organics (Cornwall U.K.),Lancaster Synthesis (Windham N.H.), Maybridge Chemical Co. Ltd.(Cornwall U.K.), Parish Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc.(Waterbury Conn.), Polyorganix (Houston Tex.), Pierce Chemical Co.(Rockford Ill.), Riedel de Haen AG (Hannover, Germany), Spectrum QualityProduct, Inc. (New Brunswick, N.J.), TCI America (Portland Oreg.), TransWorld Chemicals, Inc. (Rockville Md.), Wako Chemicals USA, Inc.(Richmond Va.), Novabiochem and Argonaut Technology.

Compounds useful for co-administration with the glutenases and treatedfoodstuffs of the invention can also be made by methods known to one ofordinary skill in the art. As used herein, “methods known to one ofordinary skill in the art” may be identified though various referencebooks and databases. Suitable reference books and treatises that detailthe synthesis of reactants useful in the preparation of compounds of thepresent invention, or provide references to articles that describe thepreparation, include for example, “Synthetic Organic Chemistry”, JohnWiley & Sons, Inc., New York; S. R. Sandler et al., “Organic FunctionalGroup Preparations,” 2nd Ed., Academic Press, New York, 1983; H. O.House, “Modern Synthetic Reactions”, 2nd Ed., W. A. Benjamin, Inc. MenloPark, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed.,John Wiley & Sons, New York, 1992; J. March, “Advanced OrganicChemistry: Reactions, Mechanisms and Structure”, 4th Ed.,Wiley-Interscience, New York, 1992. Specific and analogous reactants mayalso be identified through the indices of known chemicals prepared bythe Chemical Abstract Service of the American Chemical Society, whichare available in most public and university libraries, as well asthrough on-line databases (the American Chemical Society, Washington,D.C., www.acs.org may be contacted for more details). Chemicals that areknown but not commercially available in catalogs may be prepared bycustom chemical synthesis houses, where many of the standard chemicalsupply houses (e.g., those listed above) provide custom synthesisservices.

The glutenase proteins of the invention and/or the compoundsadministered therewith are incorporated into a variety of formulationsfor therapeutic administration. In one aspect, the agents are formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and are formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, and aerosols.As such, administration of the glutenase and/or other compounds can beachieved in various ways, usually by oral administration. The glutenaseand/or other compounds may be systemic after administration or may belocalized by virtue of the formulation, or by the use of an implant thatacts to retain the active dose at the site of implantation.

In pharmaceutical dosage forms, the glutenase and/or other compounds maybe administered in the form of their pharmaceutically acceptable salts,or they may also be used alone or in appropriate association, as well asin combination with other pharmaceutically active compounds. The agentsmay be combined, as previously described, to provide a cocktail ofactivities. The following methods and excipients are exemplary and arenot to be construed as limiting the invention.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

In one embodiment of the invention, the oral formulations compriseenteric coatings, so that the active agent is delivered to theintestinal tract. Enteric formulations are often used to protect anactive ingredient from the strongly acid contents of the stomach. Suchformulations are created by coating a solid dosage form with a film of apolymer that is insoluble in acid environments, and soluble in basicenvironments. Exemplary films are cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methylcellulose phthalate andhydroxypropyl methylcellulose acetate succinate, methacrylatecopolymers, and cellulose acetate phthalate.

Other enteric formulations comprise engineered polymer microspheres madeof biologically erodable polymers, which display strong adhesiveinteractions with gastrointestinal mucus and cellular linings and cantraverse both the mucosal absorptive epithelium and thefollicle-associated epithelium covering the lymphoid tissue of Peyer'spatches. The polymers maintain contact with intestinal epithelium forextended periods of time and actually penetrate it, through and betweencells. See, for example, Mathiowitz et al. (1997) Nature 386 (6623):410-414. Drug delivery systems can also utilize a core of superporoushydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh etal. (2001) J Control Release 71(3):307-18.

In another embodiment, a microorganism, for example bacterial or yeastculture, capable of producing glutenase is administered to a patient.Such a culture may be formulated as an enteric capsule; for example, seeU.S. Pat. No. 6,008,027, incorporated herein by reference.Alternatively, microorganisms stable to stomach acidity can beadministered in a capsule, or admixed with food preparations.

In another embodiment, the glutenase is admixed with food, or used topre-treat foodstuffs containing glutens. Glutenase present in foods canbe enzymatically active prior to or during ingestion, and may beencapsulated or otherwise treated to control the timing of activity.Alternatively, the glutenase may be encapsulated to achieve a timedrelease after ingestion, e.g. in the intestinal tract.

Formulations are typically provided in a unit dosage form, where theterm “unit dosage form,” refers to physically discrete units suitable asunitary dosages for human subjects, each unit containing a predeterminedquantity of glutenase in an amount calculated sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the unit dosageforms of the present invention depend on the particular complex employedand the effect to be achieved, and the pharmacodynamics associated witheach complex in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are commercially available. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are commercially available. Any compound useful inthe methods and compositions of the invention can be provided as apharmaceutically acceptable base addition salt. “Pharmaceuticallyacceptable base addition salt” refers to those salts which retain thebiological effectiveness and properties of the free acids, which are notbiologically or otherwise undesirable. These salts are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, thesodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like. Preferred inorganicsalts are the ammonium, sodium, potassium, calcium, and magnesium salts.Salts derived from organic bases include, but are not limited to, saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like.Particularly preferred organic bases are isopropylamine, diethylamine,ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Depending on the patient and condition being treated and on theadministration route, the glutenase may be administered in dosages of0.01 mg to 500 mg/kg body weight per day, e.g. about 20 mg/day for anaverage person. A typical dose of glutenase in patients will be in atleast about 1 mg/adult, more usually at least about 10 mg; andpreferably at least about 50 mg; usually not more than about 5 g, moreusually not more than about 1 g, and preferably not more than about 500mg. Dosages will be appropriately adjusted for pediatric formulation. Inchildren the effective dose may be lower, for example at least about 0.1mg, or 0.5 mg. In combination therapy involving, for example, a PEP+DPPIV or PEP+DCP I, a comparable dose of the two enzymes may be given;however, the ratio will be influenced by the relative stability of thetwo enzymes toward gastric and duodenal inactivation.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific enzyme, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the glutenasesare more potent than others. Preferred dosages for a given enzyme arereadily determinable by those of skill in the art by a variety of means.A preferred means is to measure the physiological potency of a givencompound.

Other formulations of interest include formulations of DNA encodingglutenases of interest, so as to target intestinal cells for geneticmodification. For example, see U.S. Pat. No. 6,258,789, hereinincorporated by reference, which discloses the genetic alteration ofintestinal epithelial cells.

The methods of the invention are used to treat foods to be consumed orthat are consumed by individuals suffering from Celiac Sprue and/ordermatitis herpetiformis by delivering an effective dose of glutenase.If the glutenase is administered directly to a human, then the activeagent(s) are contained in a pharmaceutical formulation. Alternatively,the desired effects can be obtained by incorporating glutenase into foodproducts or by administering live organisms that express glutenase, andthe like. Diagnosis of suitable patients may utilize a variety ofcriteria known to those of skill in the art. A quantitative increase inantibodies specific for gliadin, and/or tissue transglutaminase isindicative of the disease. Family histories and the presence of the HLAalleles HLA-DQ2 [DQ(a1*0501, b1*02)] and/or DQ8 [DQ(a1*0301, b1*0302)]are indicative of a susceptibility to the disease.

The therapeutic effect can be measured in terms of clinical outcome orcan be determined by immunological or biochemical tests. Suppression ofthe deleterious T-cell activity can be measured by enumeration ofreactive Th1 cells, by quantitating the release of cytokines at thesites of lesions, or using other assays for the presence of autoimmune Tcells known in the art. Alternatively, one can look for a reduction insymptoms of a disease.

Various methods for administration may be employed, preferably usingoral administration, for example with meals. The dosage of thetherapeutic formulation will vary widely, depending upon the nature ofthe disease, the frequency of administration, the manner ofadministration, the clearance of the agent from the host, and the like.The initial dose can be larger, followed by smaller maintenance doses.The dose can be administered as infrequently as weekly or biweekly, ormore often fractionated into smaller doses and administered daily, withmeals, semi-weekly, or otherwise as needed to maintain an effectivedosage level.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of the invention or to represent that the experiments below areall or the only experiments performed. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature, andthe like), but some experimental errors and deviations may be present.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Detection of Immunodominant Peptides from Gliadin and Enzymesthat Degrade them

The following examples describe the discovery and characterization of asmall number of immunodominant peptides from gliadin, which account formost of the stimulatory activity of dietary gluten on intestinal andperipheral T lymphocytes found in Celiac Sprue patients. The proteolytickinetics of these immunodominant peptides were analyzed at the smallintestinal surface. Brush border membrane vesicles from adult ratintestines were used to show that these proline-glutamine-rich peptidesare exceptionally resistant to enzymatic processing, and that dipeptidylpeptidase IV and dipeptidyl carboxypeptidase are the rate-limitingenzymes in their digestion. Supplementation of the brush border membranewith trace quantities of a bacterial prolyl endopeptidase leads to rapiddestruction of these gliadin peptides. These results provide the basisfor enzyme-mediated therapies for treating food for provision to CeliacSprue patients, and for treating such patients directly that offerdistinct advantages over the only current therapeutic option, which isstrict exclusion of gluten containing food.

To investigate the digestion of gluten, liquid chromatography coupledmass spectroscopy analysis (LC-MS-MS) was utilized to investigate thepathways and associated kinetics of hydrolysis of immunodominant gliadinpeptides treated with rat BBM preparations. Because the rodent is anexcellent small animal model for human intestinal structure andfunction, rat BBM was chosen as a suitable model system for thesestudies.

BBM fractions were prepared from rat small intestinal mucosa asdescribed in Ahnen et al. (1982) J. Biol. Chem. 257, 12129-35. Thespecific activities of the known BB peptidases were determined to be 127μU/μg for Aminopeptidase N (APN, EC 3.4.11.2), 60 μU/μg for dipeptidylpeptidase IV (DPP IV, EC 3.4.14.5), and 41 μU/μg for dipeptidylcarboxypeptidase (DCP, EC 3.4.15.1) using standard assays. No prolineaminopeptidase (EC 3.4.11.5) or prolyl endopeptidase activity (PEP, EC3.4.21.26) activity was detectable (<5 μU/μg). Alkaline phosphatase andsucrase were used as control BBM enzymes with activities of 66 μU/μg and350 U/μg, respectively.

BBM fractions were partially purified from the small intestinal mucosaof adult female rats maintained on an ad libitum diet of wheat-basedstandard rodent chow. Total protein content was determined by a modifiedmethod of Lowry with BSA as a standard. Alkaline phosphatase activitywas determined with nitrophenyl phosphate. Sucrase activity was measuredusing a coupled glucose assay. DPP IV, proline aminopeptidase and APNwere assayed continuously at 30° C. in 0.1M Tris-HCl, pH 8.0, containing1 mM of the p-nitroanilides (=8,800 M-1 cm-1) Gly-Pro-pNA, Pro-pNA orLeu-pNA, the latter in additional 1% DMSO to improve solubility. DCPactivity was measured in a 100 μl reaction as the release of hippuricacid from Hip-His-Leu. PEP activity was determined continuously with 0.4mM Z-Gly-Pro-pNA in PBS:H₂O:dioxane (8:1.2:0.8) at 30° C. One unit isthe consumption of 1 μmol substrate per minute.

DPP IV and DCP are both up-regulated by a high proline content in thediet. However, APN activity using standard substrates was found to behigher than DPP IV even when fed extreme proline rich diets. Also,although a higher DCP vs. CPP activity has been observed with the modelpeptide Z-GPLAP at saturating concentrations, a difference in Km valuescould easily account the reversed ratio measured. The amount of 100 Mwas chosen as the initial peptide concentration, because non-saturatingkinetics (kcat/Km) were considered to be physiologically more relevantthan the maximal rates of hydrolysis (kcat).

Proteolysis with the BBM preparation was investigated using the peptide(SEQ ID NO:1) QLQPFPQPQLPY, a product of chymotryptic digestion of α-9gliadin (Arentz-Hansen et al. (2000) J. Exp. Med. 191, 603-12). Thispeptide has been shown to stimulate proliferation of T cells isolatedfrom most Celiac Sprue patients, and hence is considered to possess animmunodominant epitope. It was subjected to BBM digestion, followed byLC-MS-MS analysis. A standard 50 μl digestion mixture contained 100 μMof synthetic peptide, 10 μM tryptophan and Cbz-tryptophan as internalstandards, and resuspended BBM preparations with a final protein contentof 27 ng/μl and exogenous proteins, as indicated, in phosphate bufferedsaline. After incubation at 37° C. for the indicated time, the enzymeswere inactivated by heating to 95° C. for 3 minutes. The reactionmixtures were analyzed by LC-MS (SpectraSystem, ThermoFinnigan) using aC18 reversed phase column (Vydac 218TP5215, 2.1×150 mm) withwater:acetonitrile:formic acid (0.1%):trifluoroacetic acid (0.025%) asthe mobile phase (flow: 0.2 ml/min) and a gradient of 10% acetonitrilefor 3 minutes, 10-20% for 3 minutes, 20-25% for 21 minutes followed by a95% wash. Peptide fragments in the mass range of m/z=300-2000 weredetected by electrospray ionization mass spectroscopy using a LCQ iontrap and their identities were confirmed by MSMS fragmentation patterns.

While the parent peptide (SEQ ID NO:1) QLQPFPQPQLPY disappeared with anapparent half life of 35 min, several intermediates were observed toaccumulate over prolonged periods (FIG. 1A). The MS intensities(m/z=300-2000 g/mol) and UV₂₈₀ absorbances of the parent peptides (SEQID NO:1) QLQPFPQPQLPY and (SEQ ID NO:3) PQPQLPYPQPQLPY were found todepend linearly on concentration in the range of 6-100 μM. The referencepeptides (SEQ ID NO:4) PQPQLPYPQPQLP, (SEQ ID NO:5) QLQPFPQPQLP, (SEQ IDNO:6) QPQFPQPQLPY and (SEQ ID NO:7) QPFPQPQLP were generatedindividually by limited proteolysis of the parent peptides with 10 g/mlcarboxypeptidase A (C-0261, Sigma) and/or 5.9 g/ml leucineaminopeptidase (L-5006, Sigma) for 160 min at 37° C. and analyzed byLC-MS as in FIG. 1.

Indeed, the subsequent processing of the peptide was substantiallyretarded (FIG. 1B). The identities of the major intermediates wereconfirmed by tandem MS, and suggested an unusually high degree ofstability of the (SEQ ID NO:8) PQPQLP sequence, a common motif in T cellstimulating peptides. Based on this data and the known amino acidpreferences of the BBM peptidases, the digestive breakdown of (SEQ IDNO:1) QLQPFPQPQLPY was reconstructed, as shown in the insert of FIG. 1B.The preferred pathway involves serial cleavage of the N-terminalglutamine and leucine residues by aminopeptidase N (APN), followed byremoval of the C-terminal tyrosine by carboxypeptidase P(CPP) andhydrolysis of the remaining N-terminal QP-dipeptide by DPP IV. As seenin FIG. 1B, the intermediate (SEQ ID NO:6) QPFPQPQLPY (formed by APNattack on the first two N-terminal residues) and its derivatives areincreasingly resistant to further hydrolysis. Because the high prolinecontent seemed to be a major cause for this proteolytic resistance,digestion was compared with a commercially available non-proline controlpeptide (SEQ ID NO:9) RRLIEDNEYTARG (Sigma, St. Louis, Mo.). Initialhydrolysis was much faster (t_(1/2)=10 min). More importantly, digestiveintermediates were only transiently observed and cleared completelywithin one hour, reflecting a continuing high specificity of the BBM forthe intermediate peptides.

Because the three major intermediate products (SEQ ID NO:10) QPFPQPQLPY,(SEQ ID NO:7) QPFPQPQLP, (SEQ ID NO:11) FPQPQLP) observed during BBMmediated digestion of (SEQ ID NO:1) QLQPFPQPQLPY are substrates for DPPIV, the experiment was repeated in the presence of a 6-fold excessactivity of exogenous fungal DPP IV. Whereas the relatively rapiddecrease of the parent peptide and the intermediate levels of (SEQ IDNO:5) QLQPFPQPQLP were largely unchanged, the accumulation of DPP IVsubstrates was entirely suppressed, and complete digestion was observedwithin four hours. (FIG. 1B, open bars).

To investigate the rate-limiting steps in BBM mediated digestion ofgliadin peptides from the C-terminal end, another known immunodominantpeptide derived from wheat-gliadin, (SEQ ID NO:3) PQPQLPYPQPQLPY, wasused. Although peptides with N-terminal proline residues are unlikely toform in the small intestine (none were observed during BBM digestion of(SEQ ID NO:1) QLQPFPQPQLPY, FIG. 1A), they serve as a useful model forthe analysis of C-terminal processing, because the N-terminal end ofthis peptide can be considered proteolytically inaccessible due tominimal proline aminopeptidase activity in the BBM. As shown in FIG. 2,this peptide is even more stable than (SEQ ID NO:1) QLQPFPQPQLPY. Inparticular, removal of the C-terminal tyrosine residue bycarboxypeptidase P(CPP) is the first event in its breakdown, and morethan four times slower than APN activity on (SEQ ID NO:1) QLQPFPQPQLPY(FIG. 1B). The DCP substrate (SEQ ID NO:4) PQPQLPYPQPQLP emerges as amajor intermediate following carboxypeptidase P catalysis, and is highlyresistant to further digestion, presumably due to the low level ofendogenous DCP activity naturally associated with the BBM. To confirmthe role of DCP as a rate-limiting enzyme in the C-terminal processingof immunodominant gliadin peptides, the reaction mixtures weresupplemented with rabbit lung DCP. Exogenous DCP significantly reducedthe accumulation of (SEQ ID NO:4) PQPQLPYPQPQLP after overnightincubation in a dose dependent manner. Conversely, the amount ofaccumulated (SEQ ID NO:4) PQPQLPYPQPQLP increased more than 2-fold inthe presence of 10 μM of captopril, a DCP-specific inhibitor, ascompared with unsupplemented BBM.

Together, the above results demonstrate that (i) immunodominant gliadinpeptides are exceptionally stable toward breakdown catalyzed by BBMpeptidases, and (ii) DPP IV and especially DCP are rate-limiting stepsin this breakdown process at the N- and C-terminal ends of the peptides,respectively. Because BBM exopeptidases are restricted to N- orC-terminal processing, it was investigated if generation of additionalfree peptide ends by pancreatic enzymes would accelerate digestion. Ofthe pancreatic proteases tested, only elastase at a high(non-physiological) concentration of 100 ng/μl was capable ofhydrolyzing (SEQ ID NO:3) PQPQLPYPQPQ^(↓)LPY. No proteolysis wasdetected with trypsin or chymotrypsin.

Alerted by the high proline content as a hallmark of most immunogenicgliadin peptides, a proline-specific endopeptidase was tested for thegeneration of new, free peptide termini. A literature search onavailable proteases led to the identification of prolyl endopeptidase(PEP) from Flavobacterium meningosepticum, which is specific for theC-terminal cleavage of prolines and readily available from recombinantsources (Yoshimoto et al. (1991) J. Biochem. 110, 873-8). The stable(SEQ ID NO:4) PQPQLPYPQPQLP intermediate was digested with BBM in thepresence of exogenous PEP. FIG. 3 shows the dose dependent accelerationof (SEQ ID NO:4) PQPQLPYPQPQLP digestion with increasing PEPconcentration. As little as 3.5 pg PEP/27 ng BBM protein was sufficientto double the extent of proteolysis of this gliadin fragment compared toincubation with BBM alone. In comparison, other commonly used proteaseslike papain, bromelain or porcine elastase were much less efficient,requiring 30-fold (papain) or 3000-fold (bromelain, elastase) higheramounts of enzyme compared to PEP to give similar results. Theirproteolysis was restricted to the cleavage of the GIn⁴-Leu⁵ and/orGln¹¹-Leu¹² bonds.

Prolyl endopeptidase (EC 3.4.21.26) had a preference for the Pro8-Gln9and to a lesser extent the Pro6-Tyr7 bond of the (SEQ ID NO:4)PQPQLP↓YP↓QPQLP peptide. A similar preferential cleavage was found for(SEQ ID NO:1) QLQPFP↓QPQLPY. This is in agreement with the preference ofthis prolyl endopeptidase for a second proline in the S2′ position(Bordusa and Jakubke (1998) Bioorg. Med. Chem. 6, 1775-80). Based onthis P↓XP motif and on the present data, up to 16 new, major cleavagesites can be predicted in the α2-gliadin sequence, a major source ofimmunodominant epitopes identified thus far upon PEP treatment. All ofthem are located in the critical N-terminal part. The internal cleavageby PEP can be expected to generate additional (otherwise inaccessible)substrates for DPP IV and DCP thereby complementing the naturalassimilation process of gliadins by the BBM. Thus, the specificity ofprolyl endopeptidase is ideally suited for detoxification of persistentimmunoactive gliadin peptides in Celiac Sprue.

The above data demonstrates that proline-rich gliadin peptides areextraordinarily resistant to digestion by small intestinal endo- andexopeptidases, and therefore are likely to accumulate at highconcentrations in the intestinal cavity after a gluten rich meal. Thepathological implication of digestive resistance is strengthened by theobserved close correlation of proline content and celiac toxicity asobserved in the various common cereals (Schuppan (2000) Gastroenterology119, 234-42). This analysis of the digestive pathways of immunodominantpeptides also provides a mechanism for determining whether enzymescapable of accelerating this exceptionally slow process can betherapeutically useful in the Celiac Sprue diet.

Addition of exogenous DPP IV and DCP can compensate for theintrinsically slow proline processing by the BBM, although both enzymesrely on efficient generation of free N- and C-termini by endoproteolyticcleavage. In a preferred embodiment, a soluble bacterial prolylendopeptidase (PEP) is used, which was shown to be extremely efficientat hydrolyzing the proline-rich gliadin fragments. Although PEP isexpressed in human brain, lung, kidney and intestine, no such activityhas been reported in the brush border.

Supplementation of the Celiac Sprue diet with bioavailable PEP (with orwithout DPP IV and/or DCP), by virtue of facilitating gliadin peptidecleavage to non-toxic and/or digestible fragments, is useful inattenuating or eliminating the inflammatory response to gluten. Such atreatment regimen is analogous to the enzyme therapy treatment used totreat lactose intolerance, where orally administered lactase iseffective in cleaving and thereby detoxifying the lactose in milkproducts. Prolyl endopeptidases are widely distributed inmicroorganisms, plants and animals and have been cloned from Aeromonashydrophyla (Kanatani et al. (1993) J. Biochem. 113, 790-6); Pyrococcusfurious (Robinson et al. (1995) Gene 152, 103-6) and from pig brain(Rennex et al. (1991) Biochemistry 30, 2195-2030). These isozymesconstitute alternative detoxifying peptidases. Furthermore, the prolylendopeptidase used in this study is readily amenable to proteinengineering by directed evolution. Thus, optimization of PEP specificitytowards immunogenic gliadin peptides can be achieved.

Example 2

Further Characterization of Immunodominant Gliadin Peptides and Meansfor their Digestion

It has long been known that the principal toxic components of wheatgluten are a family of closely related Pro-Gln rich proteins calledgliadins. Peptides from a short segment of α-gliadin appear to accountfor most of the gluten-specific recognition by CD4+ T cells from CeliacSprue patients. These peptides are substrates of tissue transglutaminase(tTGase), the primary auto-antigen in Celiac Sprue, and the products ofthis enzymatic reaction bind to the class II HLA DQ2 molecule. Thisexample describes a combination of in vitro and in vivo animal and humanstudies used to characterize this “immunodominant” region of α-gliadinas part of an unusually long proteolytic product generated by thedigestive process that: (a) is exceptionally resistant to furtherbreakdown by gastric, pancreatic and intestinal brush border proteases;(b) is the highest specificity substrate of human tissuetransglutaminase (tTGase) discovered to date; (c) contains at least sixoverlapping copies of epitopes known to be recognized by patient derivedT cells; (d) stimulates representative T cell clones that recognizethese epitopes with sub-micromolar efficacy; and (e) has homologs inproteins from all toxic foodgrains but no homologs in non-toxicfoodgrain proteins. In aggregate, these findings demonstrate that theonset of symptoms upon gluten exposure in the Celiac Sprue patient canbe traced back to a small segment of α-gliadin. Finally, it is shownthat this “super-antigenic” long peptide can be detoxified in vitro andin vivo by treatment with bacterial prolyl endopeptidase, providing apeptidase therapy for Celiac Sprue.

Identification of stable peptides from gastric protease, pancreaticprotease and brush border membrane peptidase catalyzed digestion ofrecombinant α2-gliadin: The protein α2-gliadin, a representativeα-gliadin (Arentz-Hansen et al. (2000) Gut 46:46), was expressed inrecombinant form and purified from E. coli. The α2-gliadin gene wascloned in pET28a plasmid (Novagen) and transformed into the expressionhost BL21(DE3) (Novagen). The transformed cells were grown in 1-litercultures of LB media containing 50 μg/ml of kanamycin at 37° C. untilthe OD600 0.6-1 was achieved. The expression of α2-gliadin protein wasinduced with the addition of 0.4 mM isopropyl β-D-thiogalactoside(Sigma) and the cultures were further incubated at 37° C. for 20 hours.The cells expressing the recombinant α2-gliadin were centrifuged at 3600rpm for 30 minutes. The pellet was resuspended in 15 ml of disruptionbuffer (200 mM sodium phosphate; 200 mM NaCl; 2.5 mM DTT; 1.5 mMbenzamidine; 2.5 mM EDTA; 2 mg/L pepstatin; 2 mg/L leupeptin; 30% v/vglycerol) and lysed by sonication (1 minute; output control set to 6).After centrifugation at 45000 g for 45 min, the supernatant wasdiscarded and the pellet containing gliadin protein was resuspended in50 ml of 7 M urea in 50 mM Tris (pH=8.0). The suspension was againcentrifuged at 45000 g for 45 min and the supernatant was harvested forpurification. The supernatant containing α2-gliadin was incubated with 1ml of nickel-nitrilotriacetic acid resin (Ni-NTA; Qiagen) overnight andthen batch-loaded on a column with 2 ml of Ni-NTA. The column was washedwith 7M urea in 50 mM Tris (pH=8.0), and α2-gliadin was eluted with 200mM imidazole, 7 M urea in 50 mM Tris (pH=4.5). The fractions containingα2-gliadin were pooled into a final concentration of 70% ethanolsolution and two volumes of 1.5M NaCl were added to precipitate theprotein. The solution was incubated at 4° C. overnight and the finalprecipitate was collected by centrifugation at 45000 g for 30 min,rinsed in water, and re-centrifuged to remove the urea. The finalpurification step of the α-2 gliadin was developed with reverse-phaseHPLC. The Ni-NTA purified protein fractions were pooled in 7 M ureabuffer and injected to a Vydac (Hesperia, Calif.) polystyrenereverse-phase column (i.d. 4.6 mm×25 cm) with the starting solvent (30%of solvent B:1:1 HPLC-grade acetonitrile/isopropanol:0.1% TFA). SolventA was an aqueous solution with 0.1% TFA. The separation gradientextended from 30-100% of solvent B over 120 min at a flow rate of 0.8ml/min.

TABLE 2 Amount of Peptides Digested after 15 hours 33-mer Control AControl B H1P0 <20% >90% >90% H2P0 <20% >61% >85% H3P0 <20% >87% >95%H4P0 <20% >96% >95% H5P0 <20% >96% >95%

The purity of the recombinant gliadin was >95%, which allowed for facileidentification and assignment of proteolytic products by LC-MS/MS/UV.Although many previous studies utilized pepsin/trypsin treated gliadins,it was found that, among gastric and pancreatic proteases, chymotrypsinplayed a major role in the breakdown of α2-gliadin, resulting in manysmall peptides from the C-terminal half of the protein and a few longer(>8 residues) peptides from the N-terminal half, the most noteworthybeing a relatively large fragment, the 33-mer (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (residues 57-89). This peptide was ofparticular interest for two reasons: (a) whereas most other relativelystable proteolytic fragments were cleaved to smaller fragments when thereaction times were extended, the 33-mer peptide remained intact despiteprolonged exposure to proteases; and (b) three distinct patient-specificT cell epitopes identified previously are present in this peptide,namely, (SEQ ID NO:20) PFPQPQLPY, (SEQ ID NO:21) PQPQLPYPQ (3 copies),and (SEQ ID NO:22) PYPQPQLPY (2 copies).

To establish the physiological relevance of this peptide, compositegastric/pancreatic enzymatic digestion of α2 gliadin was then examined.As expected, enzymatic digestion with pepsin (1:100 w/w ratio), trypsin(1:100), chymotrypsin (1:100), elastase (1:500) and carboxypeptidase(1:100) was quite efficient, leaving behind only a few peptides longerthan 9 residues (the minimum size for a peptide to show class II MHCmediated antigenicity) (FIG. 4). In addition to the above-mentioned33-mer, the peptide (SEQ ID NO:23) WQIPEQSR was also identified, and wasused as a control in many of the following studies. The stability of the33-mer peptide can also be appreciated when comparing the results of asimilar experiment using myoglobin (another common dietary protein).Under similar proteolytic conditions, myoglobin is rapidly broken downinto much smaller products. No long intermediate is observed toaccumulate.

The small intestinal brush-border membrane (BBM) enzymes are known to bevital for breaking down any remaining peptides from gastric/pancreaticdigestion into amino acids, dipeptides or tripeptides for nutritionaluptake. Therefore a comprehensive analysis of gliadin metabolism alsorequired investigations into BBM processing of gliadin peptides ofreasonable length derived from gastric and pancreatic proteasetreatment. BBM fractions were prepared from rat small intestinal mucosa.The specific activities of known BBM peptidases were verified to bewithin the previously reported range. Whereas the half-life ofdisappearance of WQIPEQSR was −60 min. in the presence of 12 ng/μl BBMprotein, the half-life of (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF digestion was >20 h. Therefore, thelatter peptide remains intact throughout the digestive process in thestomach and upper small intestine, and is poised to act as a potentialantigen for T cell proliferation and intestinal toxicity in geneticallysusceptible individuals.

Verification of proteolytic resistance of the 33-mer gliadin peptidewith brush border membrane preparations from human intestinal biopsies:to validate the conclusions reached as described in Example 1, whichdescribes studies with rat BBM preparations, in the context of humanintestinal digestion, BBM preparations were prepared from a panel ofadult human volunteers, one of whom was a Celiac Sprue patient inremission, while the rest were found to have normal intestinalhistology. (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, (SEQ IDNO:1) QLQPFPQPQLPY (an internal sequence from the 33-mer used as acontrol), WQIPEQSR and other control peptides (100 μM) were incubatedwith BBM prepared from each human biopsy (final aminopeptidase Nactivity of 13 μU/μl) at 37° C. for varying time periods. While (SEQ IDNO:1) QLQPFPQPQLPY, (SEQ ID NO:23) WQIPEQSR and other control peptideswere completely proteolyzed within 1-5 h, the long peptide remainedlargely intact for at 19 hours. These results confirm the equivalencebetween the rat and human BBM for the purpose of this study. Moreover,these results indicate that the methods, foodstuffs, and other reagentsof the invention can be used in humans not known to have Celiac Sprue toimprove digestion and reduce any ill effects of the long peptide.

Verification of proteolytic resistance of the 33-mer gliadin peptide inintact animals: The proteolytic resistance of the 33-mer gliadinpeptide, observed in vitro using BBM from rats and humans, was confirmedin vivo using a perfusion protocol in intact adult rats (Smithson andGray (1977) J. Clin. Invest. 60:665). Purified peptide solutions wereperfused through a 15-20 cm segment of jejunum in a sedated rat with aresidence time of 20 min, and the products were collected and subjectedto LC-MS analysis. Whereas >90% of (SEQ ID NO:1) QLQPFPQPQLPY wasproteolyzed in the perfusion experiment, most of the 33-mer gliadinpeptide remained intact. These results demonstrate that the 33-merpeptide is very stable as it is transported through the mammalian uppersmall intestine. The data is shown in FIG. 5.

The 33-mer gliadin peptide is an excellent substrate for tTGase, and theresulting product is a highly potent activator of patient-derived Tcells: studies have demonstrated that regiospecific deamidation ofimmunogenic gliadin peptides by tTGase increases their affinity forHLA-DQ2 as well as the potency with which they activate patient-derivedgluten-specific T cells. It has been shown that the specificity oftTGase for certain short antigenic peptides derived from gliadin ishigher than its specificity toward its physiological target site infibronectin; for example, the specificity of tTGase for the α-gliadinderived peptide (SEQ ID NO:3) PQPQLPYPQPQLPY is 5-fold higher than thatfor its target peptide sequence in fibrinogen, its natural substrate.The kinetics and regiospecificity of deamidation of the 33-mer α-gliadinpeptide identified as above were therefore measured. The k_(cat)/K_(M)was higher than that reported for any peptide studied thus far:kcat/KM=440 min-1 mM-1 for (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF compared to kcat/KM=82 min-1 mM-1 forPQPQLPY and kcat/KM=350 min-1 mM-1 for (SEQ ID NO:3) PQPQLPYPQPQLPY.

Moreover, LC-MS-MS analysis revealed that the peptide (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF was selectively deamidated by tTGaseat the underlined residues. Because tTGase activity is associated withthe brush border membrane of intestinal enterocytes, it is likely thatdietary uptake of even small quantities of wheat gluten will lead to thebuild-up of sufficient quantities of this 33-mer gliadin peptide in theintestinal lumen so as to be recognized and processed by tTGase.

Structural characteristics of the 33-mer gliadin peptide and itsnaturally occurring homologs: Sequence alignment searches using BLASTPin all non-redundant protein databases revealed several homologs(E-value<0.001) of the 33-mer gliadin peptide, shown in FIG. 6.Interestingly, foodgrain derived homologs were only found in gliadins(from wheat), hordeins (from barley) and secalins (from rye), all ofwhich have been proven to be toxic to Celiac Sprue patients. Nontoxicfoodgrain proteins, such as avenins (in oats), rice and maize, do notcontain homologous sequences to the 33-mer gliadin. In contrast, aBLASTP search with the entire α2-gliadin sequence identified foodgrainprotein homologs from both toxic and nontoxic proteins. Based onavailable information regarding the substrate specificities of gastric,pancreatic and BBM proteases and peptidases, it is believed that,although most gluten homologs to the 33-mer gliadin peptide containedmultiple proteolytic sites and are therefore unlikely to be completelystable toward digestion, several sequences from wheat, rye and barleyare expected to be resistant to gastric and intestinal proteolysis. Thestable peptide homologs to the 33-mer α2-gliadin peptide are (SEQ IDNO:24) QPQPFPPQLPYPQTQPFPPQQPYPQPQPQYPQPQ (from α1- and a6-gliadins);(SEQ ID NO:25) QQQPFPQQPIPQQPQPYPQQPQPYPQQPFPPQQPF (from B1 hordein);(SEQ ID NO:26) QPFPQPQQTFPQQPQLPFPQQPQQPFPQPQ (from γ-gliadin); (SEQ IDNO:27) QPFPQPQQPTPIQPQQPFPQRPQQPFPQPQ (from ω-secalin). These stablepeptides are all located at the N-terminal region of the correspondingproteins. The presence of proline residues after otherwise cleavableresidues in these peptides would contribute to their proteolyticstability.

Bacterial Prolyl Endopeptidase Rapidly Detoxifies the 33-Mer GliadinPeptide:

The abundance and location of proline residues is a crucial factorcontributing to the resistance the 33-mer gliadin peptide towardgastrointestinal breakdown. In accordance with the methods of theinvention, a prolyl endopeptidase can catalyze breakdown of thispeptide, thereby diminishing its toxic effects. Preliminary in vitrostudies with short gliadin peptides and the prolyl endopeptidase (PEP)from F. meningosepticum demonstrate this aspect of the invention. Theability of this PEP to clear the 33-mer gliadin peptide was evaluatedvia in vitro and in vivo experiments. Using both rat BBM andco-perfusion of the peptide and PEP in intact rat intestines, thisdetoxification was demonstrated. The results are shown in FIG. 7.Together these results highlight the potential of detoxifying gluten inCeliac Sprue patients by peptidase therapy.

Although gluten proteins from foodgrains such as wheat, rye and barleyare central components of a nutritious diet, they can be extremely toxicfor patients suffering from Celiac Sprue. To elucidate the structuralbasis of gluten toxicity in Celiac Sprue, comprehensive proteolyticanalysis was performed on a representative recombinant gliadin underphysiologically relevant conditions. An unusually long andproteolytically stable peptide product was discovered, whosephysiological relevance was confirmed by studies involving brush bordermembrane proteins from rat and human intestines as well as intestinalperfusion assays in live rats. In aggregate, these data demonstrate thatthis peptide and its homologs found in other wheat, rye and barleyproteins contribute significantly to the inflammatory response todietary wheat in Celiac Sprue patients.

The absence of satisfactory animal models for Celiac Sprue implies thatthe pivotal pathogenic nature of the gluten peptides identified in thisstudy can only be verified in human patients. While this is likely to bea formidable task, and would in any event need to be conducted in amanner that would not harm the patient, the results above demonstratethat the deleterious effects of gluten ingestion by Celiac Spruepatients can be amelioriated by enzyme treatment of gluten containingfoods. Specifically, co-administration of a bioavailable form of asuitable prolyl endopeptidase with dietary gluten would attenuate itstoxicity by cleaving the stable 33-mer peptide into non-immunogenicproducts. Given the absence of a satisfactory therapeutic option forCeliac Sprue and the notorious difficulty associated with long-termmaintenance of a gluten-free diet, the peptidase therapies of thepresent invention provides an alternative to strict abstinence for therapidly growing numbers of individuals affected by this disease.

Example 3 Peptidase Supplementation as Therapy for Celiac SprueDemonstration of Efficacy and Safety in Rats and Humans In Vivo

As described above, Celiac Sprue is a disease engendered by the gliadinpeptides in wheat, rye, or barley that interact with the small intestineto produce a cascade of events leading to the destruction of intestinalmucosa and consequent malabsorption of nutrients and vitamins. Gliadinpeptides are highly resistant to digestion by gastric, and pancreaticproteases as well as by the integral peptidases of the intestinal brushborder surface. The interaction of recombinant α-gliadin with mammalianpepsin, chymotrypsin, trypsin, and elastase has shown that a 33-residuepeptide rich in glutamine(Q) and proline(P) residues ((SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) is a major final digestion product.When this 33-mer is exposed to intact rat small intestine or to humanintestinal brush border membranes, it is impervious to additionalbreakdown. This peptide has very high specificity for stimulating T cellproliferation in peripheral blood lymphocyte cultures from Celiac Spruepatients but not in lymphocyte cultures from normal individuals. Becauseof the resistance of this and other gliadin peptides to pancreatic andintestinal digestion and the abundance of proline residues in thesepeptides, the 33-mer and other gliadin peptides were exposed to a prolylendopeptidase, which demonstrated that this enzyme is highly effectiveunder physiologic conditions in breaking the peptide bonds between theproline and the next residue on the peptide chain. The consequentcleavage of the gliadin peptide rendered it incapable of inducinglymphocyte proliferation, demonstrating that the additional processingof the gliadin peptide should prevent its toxic reaction to theintestine in Celiac Sprue patients.

This example describes experiments to demonstrate that a PEP iseffective in further digesting the toxic gliadin peptides underphysiological conditions. Celiac Sprue patients, in accordance with themethods of the present invention, can consume a normal diet along with aPEP supplement that will digest the toxic gliadin peptide and circumventthe reaction that leads to T-cell proliferation and destruction of theintestinal mucosa. This is an alternative and innovative treatment inCeliac Sprue—the substitution of a rigid gluten-free diet with anexogenous digestive endopeptidase that promotes metabolism and essentialdetoxification of the gliadin peptide. The studies described in thisExample can be used to document efficacy and safety and include a pilotstudy in controls and Celiac patients with PEP-treated wheat flour.These are important studies that enable the institution of a fullclinical trial in normal humans and those with Celiac Sprue.

The studies described in this example include the following:

1. To determine whether exogenous peptidase supplementation digestsresistant gliadin peptides to non-toxic, absorbable products in the ratin vivo under physiologic conditions. This study involves:

A. Expression and purification of recombinant Prolyl Endopeptidase(rPEP);

B. Examination of rPEP action on gliadin peptides in rat intestine invivo; and determination of optimal conditions for efficient digestionand analysis of effects on intestinal structure and function with acuteand chronic administration.

2. To perform preliminary clinical testing of the efficacy of PEP inprocessing the resistant gliadin peptides in wheat flour to non-toxicproducts.

3. To establish the ideal conditions for packaging the rPEP to achieveefficient digestion of gliadin peptides in vivo, including thepreparation of formulations of rPEP (polyanhyride capsules,methacrylate-glycol capsules; OROS).

As described in the preceding examples, experiments involving exposureof α-gliadin to purified pancreatic proteases have demonstrated theproduction of a 33-residue glutamine and proline-rich peptide ((SEQ IDNO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) as a major end product. Whenthis peptide is administered by perfusion into the small intestine ofthe intact rat under physiologic conditions or incubated with humanintestinal brush border membranes, its digestion is relatively retardedas compared to that for most dietary peptides such as myoglobin fromperipheral muscle. Experiments demonstrate that a prolyl endopeptidase(PEP) (from Flavobacterium meningosepticum) at molar concentrations onlyone one-hundredth of those of the digestive resistant 33-mer gliadinpeptide is capable of efficiently cleaving it to smaller peptides thatare 1) non-toxic residual peptides (as estimated from the human T cellproliferation assay), and 2) can be readily further digested andabsorbed by the rat intestine. The conditions for optimal action of thePEP on the resistant α-gliadin 33-mer peptide and other gliadin peptidesthat react in the T cell proliferation assay can be determined by themethods set forth in this example.

To demonstrate that exogenous peptidase supplementation digestsresistant gliadin peptides to non-toxic, absorbable products in vivounder physiologic conditions, expression and purification of recombinantProlyl Endopeptidase (rPEP) can be undertaken. Recombinant prolylendopeptidase (rPEP) from Flavobacterium meningosepticum can beconstructed and expressed as detailed by Yoshimoto Tet et al., and byUchiyama et al. One can also obtain recombinant preparations of a PEPenzyme from Aeromonas Hydrophila as detailed by Shen et al. or fromSphingomonas capsulate (Kabashima T, Fujii M, Meng Y, Ito K, YoshimotoT., Prolyl endopeptidase from Sphingomonas capsulate: isolation andcharacterization of the enzyme and nucleotide sequence of the gene, ArchBiochem Biophys. 1998 Oct 1; 358(1):141-8.)

To demonstrate rPEP action on gliadin peptides in rat intestine in vivoand to determine optimal conditions for efficient digestion, intestinalperfusion studies in intact rats can be performed as follows.Sprague-Dawley rats (300-400 gms) are anesthetized with pentobarbital,the abdomen entered through a midline incision, and a 10-20-cm length ofjejunum isolated and catheterized as detailed previously. A test peptide(1 mM GLGG) known to be digested efficiently at the intestinal surfaceis perfused through the isolated segment at 0.4 ml per min in 154 mMNaCl/0.1% polyethylene glycol to allow calculation for any water flux.The disappearance of the test peptide and the appearance of any productswill allow calculation of the intestinal surface digestion andabsorption. These results are compared with those with the digestiveresistant gliadin peptides (SEQ ID NO:1) QLQPFPQPQLPY, (SEQ ID NO:3)PQPQLPYPQPQLPY, and the 33-mer (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. The GLGG peptide is readilyhydrolyzed to free Leu and Gly and to the dipeptide GG; the high prolinecontent of the gliadin peptides makes them poor substrates for theavailable intestinal membrane peptidases. The intestinal luminal samplestaken at the site of the distal catheter are analyzed by LiquidChromatography-Mass Spectrometry (SpectraSystem, ThermoFinnigan) on aC18 reversed phase column, as previously detailed. Peptide fragments aredetected and their identities confirmed by mass spectrometryfragmentation patterns under conditions where there is a linearrelationship of these peptides and their products.

After the relative degree of digestion and absorption of the GLGG andgliadin peptides has been established, experiments to demonstrate theefficacy of PEP in digesting the peptides in this in vivo rat model canbe performed. Initially, the PEP is perfused via a separate catheter atthe proximal infusion site of the isolated jejunal segment at molarconcentrations ranging from 1:1000 to 1:1 of that of the test peptides.Preliminary experiments show that a molar ratio of PEP:peptide of 1:100is sufficient for efficient cleavage at the C-terminus of internalProlyl residues to the gliadin sequence. But, it is also important totest the PEP at higher concentrations, in case more peptidase activityis required and desired for total cleavage of the gladin peptides and toassess side effects on the integrity of the intestine and other organs.After each experiment, the intestine and other abdominal organs(especially liver and kidney) are recovered, aliquots quick frozen andpreserved at −70° for subsequent assay of intestinal carbohydrases(sucrase, lactase, maltase) and peptidases (aminopeptidase N,carboxypeptidase, dipeptidyl peptidase IV), and the tissues are fixed informalin, stained for hematoxylin-eosin, and examined (“blindly” withoutknowledge of the experimental protocol) for any histological changes,with particular attention paid to any structural effects that might beproduced of the higher PEP concentrations.

Once the ideal ratio of PEP to gliadin peptide is determined in theseperfusion experiments, one can analyze the capacity of the PEP toenhance the hydrolysis of gluten peptides in commercialgluten-containing wheat flour. A 1% slurry of the flour mixed with 1:100(weight basis) trypsin and chymotrypsin, and 1:500 (weight basis)elastase is perfused into the intestine with or without co-perfusion ofsuitable quantities of the PEP. LC-MS analysis of the residual gliadinproducts is conducted on the collected samples, and the histologic andenzymatic parameters are examined, as described above.

Feeding studies in intact rats can be conducted as follows. Once theideal ratio of the PEP to the gliadin substrate has been established inthe perfusion experiments, rats are fed 70% carbohydrate chow containingwheat flour, which is used as the conventional rat chow for periods oftwo weeks. Control rats are fed only the special chow, and the treatedrats are given sufficient PEP supplementation (molar ratios PEP togliadin protein: 1:1, 1:10 and 1:100) in the diet to digest the residualgliadin peptides such as the Pro- and Gln-rich 33-mer. After two weeksof ab lib feeding, the rats' daily consumption of food is quantified bydaily weighing of the residual chow in the feeder and the nutritionalassessment determined by daily body weights. Over the feeding period of2-4 weeks, rats are weighed and examined daily to verify normalactivities and are then killed by stunning and decapitation. Theintestine, liver and kidneys are recovered and examined for gross andhistological integrity, and any anatomic differences are noted betweenthe control (PEP−) and treated (PEP+) animals. In addition, digestiveenzymes (carbohydrases and proteases) are determined, as detailed forthe rat perfusion studies.

Preliminary clinical testing of the efficacy of PEP in processing theresistant gliadin peptides can be conducted as follows. Now that it hasbeen established that PEP can readily convert the high-Pro, high-Glngliadin peptides to smaller, non-toxic fragments that do not produceproliferation in the T cell assay, preliminary testing of PEP treatedwheat flour containing the usual or enhanced amounts of gluten (e.g.,Bob's RedMill flour, Milwaukee, Oreg.) or food-grade gluten or gliadinitself (e.g. gliadin from Sigma Aldrich) can be undertaken. The flour,gluten or gliadin can be batch treated with appropriate amounts ofpurified mixtures of pancreatic enzymes that are used clinically totreat pancreatic insufficiency (e.g., Pancrease MT 20 containing 20,000units lipase, 44000 units of the pancreatic proteases and 56000 units ofamylase per capsule). Incubation of a slurry of flour, gluten or gliadinwith the material from an appropriate amount of capsules of thePancrease preparation can be carried out in 0.02 M Na—K phosphatebuffer, pH 6.5 at 37° C. for several hours under sterile conditionsuntil 1) standard T cell proliferation assays (see, for example,Arentz-Hansen, 2000) identifies the highly active signal produced by thegliadin peptides and particularly 33-mer, ((SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) and 2) the average size of theproteolytically derived gliadin peptides has been reduced to <6 residues(as measured by gel filtration HPLC). The pre-digested flour, gluten orgliadin is then exposed to sufficient pure PEP (for example, 1 molePEP:100 moles gliadin substrate) under sterile conditons for 1-18 hours,and the cleavage of gliadin peptides with known toxicity such as the33-mer verified by LC-MS analysis. In parallel control studies,previously denatured PEP (by heating to 90° C. for 60 min) can beincubated with the protease-treated flour and the persistence of thesetoxic peptides is verified by LC-MS analysis. These tests demonstratethe usefulness of a the 33-mer in assays; in one aspect, the presentinvention provides the 33-mer in isolated and purified forms, as well asassays to detect its present in foodstuffs.

The pre-treated flours can be incorporated into otherwise gluten-freebreakfast muffins by a nutritionist, and these served to volunteerpersons and those with biopsy-proven Celiac Sprue at a “community”breakfast in the nutrition department for a period of two weeks.Patients must have uncomplicated Celiac Sprue that is in remission ongluten exclusion alone. Control volunteers who have been established notto have Celiac Sprue and negative Celiac antibody studies are alsorecruited. During this period the control muffins made with flour thathas been treated with denatured pancreatic proteases±PEP are given. ThePEP+ muffins are given for the first two weeks followed by a two weekbreak from the breakfasts, and the PEP− muffins are administered overthe second two week breakfast sessions. The study can be single-blinded,the subjects being unaware of whether PEP is included in the study. Thephysician and nutritionist will know the flour has been exposed only tothe pancreatic proteases or also to PEP, in case there are any untowardreactions to the PEP material. All study subjects will fill out aquestionnaire regarding their observations during each two week periodas well as during the two week break time and the two weeks after thesecond muffin breakfast period. Although obtaining a biopsy viaendoscopy would be an ideal monitor of the PEP efficacy, this cannot beethically justified based on currently available data. Endoscopy may beoffered only if needed as an aspect of patient care. Participants willinitially meet briefly with the responsible physician-investigator whowill be available throughout the study. Participants will be interviewedand the questionnaire reviewed by a nutritionist and physician beforethe study, at the end of each two week period and two weeks aftercompleting the study. The principal investigator will be ultimatelyresponsible for the conduct of the trial and will meet regularly withthe responsible physician and nutritionist to whom the day to dayaspects of the study will be delegated. Adults from age 17 and older canbe eligible for the study. Both males and females with Celiac Sprue willbe recruited through Celiac support organizations. Individuals fromvarious ethnic groups, including Asian and African American can berecruited, although most patients with Celiac Sprue are Caucasians. Bothmales and females can participate; there is a somewhat higher proportionof female Celiac Sprue patients (˜65%). Participants will have 24 houraccess to the gastroenterology team, and a member of the research teamwill be available for consultation. Efficacy will be monitored by thecomparative responses of participants during the control period wheningesting protease-treated flour without the PEP versus the same flourthat has been treated with PEP.

Suitable conditions for packaging the rPEP to achieve efficientdigestion of gliadin peptides in vivo can be determined as follows. Todevelop a palatable preparation of PEP to enable the in vivo digestionof the toxic peptides in humans, it can be useful to formulate PEP sothat it can pass into the small intestine without being destroyed by theharsh acidic environment of the stomach. In addition, this formulationcan provide rapid release of PEP upon entry into the duodenum, where thesecreted pancreatic proteases exert their maximal action within theluminal contents to cleave dietary proteins. There are severalwell-studied and widely used examples of such delivery systems for othersubstances. The development of an optimized formulation for an effectivePEP drug capable of delivering pharmacologically useful quantities ofthis enzyme into the upper small intestine as a digestive supplement canbe conducted as follows. To process the digestive-resistant gliadinpeptides, selected formulation strategies that have been usedsuccessfully for the delivery of other enzyme supplements can be used.In particular, previously used formulations for pancreatic proteases andlactase are evaluated by use of recombinant PEP from Flavobacteriummeningoscepticum and Aeromonas hydrophila. These enzymes are expressedand purified as described by A. Kitazono et al. and A. Kanatani et al.Pancreatic enzymes have been used for the past seventy years to treatpancreatic exocrine insufficiency. Although early clinical results werevariable due to gastric inactivation of the exogenously administeredenzymes, a revived interest in enzyme-containing digestive aids occurredaround 1960 with the development of acid stable enteric coatings (I. R.Wilding, S. S. Davis, and D. T. O'Hagan, Targeting of drugs and vaccinesto the gut. Pharmac. Ther. 62, 97-124, (1994)). Similarly, acid stableenteric coatings have also been used for the delivery of lactase intothe duodenum of patients with lactase deficiency. In one embodiment, theglutenase formulations of the invention comprise a glutenase in a stableenteric coating.

Lyophilized, particulate PEP mixed with bicarbonate (as buffer) iscoated with Eudragit S100, L30D or L 100-44 according to manufacturer'sinstructions (Rohm America). Alternatively, cellulose acetate phthalate,methylcellulose or hydroxypropylmethyl cellulose phthalate can be usedas coatings for the preparation of gastric acid resistant pellets. Theseenteric coatings are commonly used for the formulation of pancreatin(see T. Sipos (1978), Preparation of enteric coated digestive enzymecompositions, U.S. Pat. No. 4,079,125; and T. Sipos (1998), Highbuffer-containing enteric coating digestive enzyme bile acidcompositions and method of treating digestive disorders therewith, U.S.Pat. No. 5,750,104).

An alternative strategy useful in preparing formulations of theinvention, used successfully with lactase (B. J. Langner (1999), Entericpolymer coated capsule containing dried bacterial culture for supplyinglactase, U.S. Pat. No. 6,008,027), involves filling gelatin capsuleswith 50-90% lyophilized PEP, the remaining capacity being filled withstabilizing dessicants such as silicon oxide, silicon dioxide ormicrocrystalline cellulose and bicarbonate buffer. The capsules areenterically coated with Eudragit polymer (Rohm America) or polyvinylacetate phthalate (Sureteric, Merck Frosst) and vacuum dried prior touse. Similarly, diastase has been formulated with Eudragits RS100 andcellulase acetate phthalate coatings for enteric use (S. P. Vyas, P. J.Gogoi, S. Pande, and V. K. Dixit, Enteric spherules diastase in enzymepreparations. J. Microencapsulation. 8, 447-454, 1991). To demonstratethat these or other formulations increase PEP bioavailability in thesmall intestine, one can perform the following experiments. First, theability of PEP activity to withstand 0.5-2 h of simulated gastrictreatment (pepsin, in 0.1N HCl, pH 2) can be evaluated. If >10% activitycan be reproducibly retained, the formulation is exposed to simulatedconditions in the duodenum (pH 6.5 buffer containing trypsin,chymotrypsin and carboxypeptidase at a 1:100 molar ratio and elastase ata 1:500 ratio to the putative α2-gliadin). Ideally, full release of PEPactivity would be achieved within 15 minutes. Formulations that satisfythe above criteria are fed initially to adult rats in conjunction withgluten-free meals spiked with recombinant α2-gliadin (whose proteolyticbehavior in response to gastric and pancreatic enzymes+PEP has been wellcharacterized). PEP doses in the range of 10-1000 units/kg body weightcan be evaluated. Animals are sacrificed two hours after meals, and thesmall intestinal derived contents are analyzed by LC-MS for residual PEPactivity and the extent to which gliadin has been proteolyzed. Inparticular, the concentration of the 33-mer digestive-resistant gliadinpeptide is estimated. Formulations that yield >90% reduction inconcentration of this peptide are evaluated more extensively forpotential toxicity, as detailed above for the initial rat studies withwater soluble PEP.

The procedures described herein are performed under an approved AnimalProtocol described below. Male Sprague-Dawley rats, 250-300 g, (orFisher rats for studies of DPP IV deficient intestine) are allowedaccess to regular wheat-based rat chow until the experiment. Rats areallowed water only for 8 hours prior to the experiment to insureclearance of residual chow in the upper small intestine. After the ratis anesthetized with an intraperitoneal injection of pentobarbital (50mg/Kg), the abdominal cavity is opened and a small incision made in asegment of jejunum located 10 cm beyond the ligament of Trietz.Cannulation is made with a polyethylene catheter (3 mm ID, 4 mm OD) andsutured 2 cm distal to the incision. A second cannula is placed insimilar fashion 10 cm distal to the first with the cannula facingproximally. After rinsing the isolated, intact jejunal segment withRingers solution (140 mM NaCl, 10 mM KHCO₃, 1.2 mM K₂HPO₄, 1.2 mM CaCl₂,1.2 mM MgCl₂) at 37° C. to remove any intraluminal debris, the isolatedloop of intestine is returned to the abdominal cavity. The incision iscovered with clear plastic wrap, and intra-abdominal temperaturemaintained at 37° C. by positioning a 30 watt incandescent lap at ˜30 cmfrom the animal. A 2 mM solution of a gliadin peptide of 7-14 residues(purified and characterized by HPLC-Mass Spectrometry) is perfused inRinger's solution containing [¹⁴C]inulin (a dilution-concentrationmarker) to establish a steady state of concentrations of residualpeptide and smaller products at the collection distal collection site(previous studies with other peptides and carbohydrates have revealedthe steady state to be achieved in 10-20 minutes). Samples collected atthe distal site are recovered and analyzed by HPLC-MS for residualpeptide and smaller peptide or amino acid products. Samples arecollected over 3 successive 10 minute periods after a steady state isachieved, and a series of gliadin and non-gliadin peptides are used.Animals can usually be maintained under anesthesia for a period of 3 to6 hours by the addition of small increments of pentobarbital (˜5 mg per30-60 minutes). At the end of the experiment, the intestinal segment andan adjacent control segment are recovered and samples taken from liver,kidney and blood for analysis of the test peptide and its products.Terminal euthanasia is accomplished by an overdose of anesthesia toproduce apnea until there is no heart contraction.

While other methods and reagents can be employed for purposes of thepresent invention, this example provides enzymes, enzyme formulations,and animal and clinical testing protocols to demonstrate the efficacy ofenzyme-mediated therapy for Celiac Sprue.

Example 4 Heterologous Expression of PEP in Lactobacilli

In one embodiment of the present invention, a Celiac Sprue patient isprovided with a recombinant organism modified to express a PEP of theinvention. The recombinant organism is selected from those organismsthat can colonize the intestinal mucosa without detriment to thepatient, thereby providing an endogenous source of PEP to the patient.As one example, Lactobacilli such as L. casei and L. plantarium cancolonize the intestinal mucosa and secrete PEP enzymes locally. Giventheir widespread use in food processing, they can also be used as anefficient source of PEP for industrial (to treat foodstuffs) and medical(to prepare PEP for pharmaceutical formulation) use. PEPs can beexpressed in such lactobacilli using standard recombinant DNAtechnologies. For example, Shaw et al. (Shaw, D M, Gaerthe, B; Leer, RJ, Van der Stap, J G M M, Smittenaar, C.; Den Bak-Glashouwer, Heijne, MJ, Thole, J E R, Tielen F J, Pouwels, P H, Havenith, C E G (2000)Immunology 100, 510-518) have engineered Lactobacilli species to expressintracellular and surface-bound tetanus toxin. The intact PEP genes(including leader sequences for efficient bacterial secretion) can becloned into shuttle expression vectors such as pLP401 or pLP503 undercontrol of the (regulatable) amylase promoter or (constitutive) lactatedehydrogenase promoter, respectively. Alternatively, recombinant foodgrade Lactobacilli strains can be generated by site specificrecombination technology (e.g. see. Martin M C, Alonso, J C, Suarez J E,and Alvarez M A Appl. Env. Microbiol. 66, 2599-2604, 2000). Standardcultivation conditions are used for Lactobacilli fermentation, such asthose described by Martin et al.

Example 5 Heterologous Expression of PEP in Yeasts

Both naturally occurring and recombinant cells and organisms can be usedto produce the glutenases useful in practice of the present invention.Preferred glutenases and producing cells include those from organismsknown to be Generally Regarded as Safe, such as Flavobacterium,Aeromonas, Sphingomonas, Lactobacillus, Aspergillus, Xanthomonas,Pyrococcus, Bacillus and Streptomyces. Extracellular glutenase enzymesmay be obtained from microorganisms such as Aspergillus oryzae andLactobacillus casei. Preferred cells include those that are already usedin the preparation of foodstuffs but have been modified to express aglutenase useful in the practice of the present invention. As oneexample, yeast strains such as Saccharomyces cerevisiae are useful forhigh level expression of secreted heterologous proteins. Genes encodingany of the PEPs described above (mature protein only) can be cloned inexpression plasmids designed for optimal production of secretedproteins. An example of such a heterologous expression strategy isdescribed in Parekh, R. N. and Wittrup, K. D. (Biotechnol. Prog. 13,117-122, 1997). Either self-replicating (e.g. 2 micron) or integrating(e.g. pAUR101) vectors can be used. The GAL1-10 promoter is an exampleof an inducible promoter, whereas the ADH2 promoter is an example of aconstitutive promoter. The cDNA encoding the mature PEP is fuseddownstream of a leader sequence containing a synthetic pre-pro regionthat includes a signal cleavage site and a Kex2p cleavage site. S.cerevisiae BJ5464 can be used as a host for production of the peptidase.Shake-flask fermentation conditions are described by Parekh and Wittrupin the above-cited reference. Alternatively, high cell density fed-batchcultures can be used for large scale production of the peptidases; arepresentative procedure for this purpose is described in Calado, C. R.C, Mannesse, M., Egmond, M., Cabral, J. M. S, and Fonseca, L. P.(Biotechnol. Bioeng. 78, 692-698, 2002).

Example 6 Enteric Capsule Formulation of Prolyl Endopeptidase

Gelatin capsules are filled with 100 mg prolyl endopeptidase and 10 mgof silicon dioxide. The capsules are enterically coated with Eudragitpolymer and put in a vacuum chamber for 72 hours. The capsules are thenheld at a range of temperature of 10° C. to 37° C. and a controlledhumidity level of 35-40%.

Example 7 Studies of Enteric Capsule Formulation of Prolyl Endopeptidase

A study is conducted where patients with Celiac Sprue are enrolled in atwo week-long study. Gelatin capsules containing 90% prolylendopeptidase mixed with 10% silicon dioxide are used. The capsules arehand-filled with the mixture, banded, and coated with a 10% Suretericenteric coating (a polymer of polyvinylacetatephthalate developed by theCanadian subsidiary of Merck & Company). Samples are acid-tested byexposing the coating to 1N HCL for one hour in order to simulate theacid environment of the stomach. The capsules are then put in a vacuumchamber for 72 hours.

Two 100 mg capsules are administered to each patient prior to each meal.The patients are instructed to eat all kinds of food without abstainingfrom those that were known to cause distress, e.g., bloating, diarrhea,and cramps.

Example 8 Enteric Pill Formulation of Prolyl Endopeptidase

400 mg of L-tartaric acid and 40 mg of polyethylene glycol-hydrogenatedcastor oil (HCO-60) are dissolved in 5 ml of methanol. This solution isplaced in a mortar previously warmed to 30° C. To the solution is added100 mg of prolyl endopeptidase. Immediately after the addition of PEP,the mixture is stirred with a pestle under a hot air current (40° C.)and then placed in a desiccator under vacuum overnight to remove thesolvent. The resulting solid-mass is pulverized with a pestle andkneaded with 30 mg of sodium bicarbonate and a small amount of 70%ethanol. The mixture is then divided and shaped into pills of about 2 mmsize and thoroughly dried. The dried pills are given a coating ofhydroxypropylmethylcellulose phthalate (HP-55) to obtain an entericformulation.

Example 9 Diagnostic Methods

The 33-mer peptide ((SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLP YPQPQPF)and its deamidated derivatives that are formed by the action of tissuetransglutaminase are useful diagnostic reagents for the detection ofCeliac Sprue. The enzyme tTGase deamidates the 33-mer at least at theunderlined positions shown in the following sequence: (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, and deamidated counterparts of the33-mer are important reagents of the present invention. Such deamidatedcounterparts may comprise one, two or more deamidated glutamine (Q)residues.

Oligoeptide analogs of the oligopeptides described by amino acidsequence herein are also included. Such analogs contain at least onedifference in amino acid sequence between the analog and nativeantigenic peptide. An L-amino acid from the native peptide may bealtered to any other one of the 20 L-amino acids commonly found inproteins, any one of the corresponding D-amino acids, rare amino acids,such as 4-hydroxyproline, and hydroxylysine, or a non-protein aminoacid, such as β-alanine and homoserine. Also included with the scope ofthe present invention are amino acids that have been altered by chemicalmeans such as methylation (e.g., α-methylvaline), deamidation, amidationof the C-terminal amino acid by an alkylamine such as ethylamine,ethanolamine, and ethylene diamine, and acylation or methylation of anamino acid side chain function (e.g., acylation of the epsilon aminogroup of lysine), deimination of arginine to citrulline,isoaspartylation, or phosphorylation on serine, threonine, tyrosine orhistidine residues. Candidate oligopeptide analogs may be screened forutility in a diagnostic method of the invention by an assay measuringcompetitive binding to antibodies, T cell receptor, etc. Those analogsthat inhibit binding of the native peptides are useful diagnosticreagents. Oligopeptides and oligopeptide analogs may be synthesized bystandard chemistry techniques, including synthesis by automatedprocedure.

Monoclonal antibodies are provided by the invention, which reactspecifically with this peptide and its deamidated derivatives. Methodsof producing antibodies are well known in the art. Polyclonal antibodiesare raised by a standard protocol, for example by injecting a productionanimal with an antigenic composition, formulated as described above.See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988. When a peptide immunogen is utilized, it isadvantageous to conjugate the peptide with a larger molecule to make animmunostimulatory conjugate. Commonly utilized conjugate proteins thatare commercially available for such use include bovine serum albumin(BSA) and keyhole limpet hemocyanin (KLH). Polyclonal antibodiesspecific for the polypeptide may then be purified from such antisera by,for example, affinity chromatography using the polypeptide coupled to asuitable solid support. Alternatively, for monoclonal antibodies,hybridomas may be formed by isolating the stimulated immune cells, suchas those from the spleen of the inoculated animal. These cells are thenfused to immortalized cells, such as myeloma cells or transformed cells,which are capable of replicating indefinitely in cell culture, therebyproducing an immortal, immunoglobulin-secreting cell line. Many suchcell lines (such as myelomas) are known to those skilled in the art. Inaddition, antibodies or antigen binding fragments may be produced bygenetic engineering. In this technique, as with the standard hybridomaprocedure, antibody-producing cells are sensitized to the desiredantigen or immunogen. The messenger RNA isolated from the immune spleencells or hybridomas is used as a template to make cDNA using PCRamplification. A library of vectors, each containing one heavy chaingene and one light chain gene retaining the initial antigen specificity,is produced by insertion of appropriate sections of the amplifiedimmunoglobulin cDNA into the expression vectors. A combinatorial libraryis constructed by combining the heavy chain gene library with the lightchain gene library. This results in a library of clones which co-expressa heavy and light chain (resembling the Fab fragment or antigen bindingfragment of an antibody molecule). The vectors that carry these genesare co-transfected into a host (e.g. bacteria, insect cells, mammaliancells, or other suitable protein production host cell). When antibodygene synthesis is induced in the transfected host, the heavy and lightchain proteins self-assemble to produce active antibodies that can bedetected by screening with the antigen or immunogen.

The 33mer peptide is exceptionally resistant toward gastrointestinalproteolysis, thereby allowing the peptide to persist as it travelsthrough the intestinal tract. Also, this peptide includes multiplecopies of immunogenic epitopes from gliadin that are recognized byantibodies in most Celiac patients. Because multivalent epitopes areknown to elicit an especially vigorous immune response (e.g. Boniface etal., 1998, Immunity 9: 459), the 33-mer and its deamidated derivativeshave inflammatory properties in the Celiac intestine, even at low doses.Moreover, as tTGase is known to become transiently linked to itssubstrate, the present invention provides fusion proteins in which allor a portion of a mammalian tTGase, including but not limited to human,bovine, equine, and porcine tTGase, is linked, usually covalently, tothe 33-mer of the invention, wherein the linkage site is at a site foreventual deamidation. This fusion protein of the invention is a highlypotent stimulator of T cells from Celiac Sprue patients in that thefusion protein exactly mimics the complexes formed in Celiac Spruepatients and is recognized by the anti-tTGase antibodies and by T cellsin those patients.

In one embodiment, the present invention provides a diagnostic forCeliac Sprue that is a urine test. It is well known that thepermeability of the small intestine increases during active Celiac Sprueand reduces again when a strict gluten-free diet is followed (e.g.Johnston et al., 2001, Lancet 358: 259). As the 33-mer peptide traversesthe small intestine, a small amount of the peptide derived from a testmeal will induce leakiness, and in turn be transported across theepithelial layer, and passed into urine. Given its proteolyticresistance, this peptide will emerge in the urine, and can be detectedby standard analytical procedures such as LC-tandem mass spectrometry oran antibody-based diagnostic test. Presence of the peptide in the urineis diagnostic for Celiac Sprue. The sensitivity of this diagnosticprocedure could be increased through the use of ¹³C or other labeledpeptide. Moreover, in current practice, an individual suspected ofhaving Celiac Sprue is typically placed on a gluten-free diet and thenchallenged with gluten some weeks later to see if symptoms reappear. Thediagnostic tests of the present invention can be used upon thephysician's first suspicion that an individual is suffering from CeliacSprue, thereby avoiding the harmful effects of placing that individualback on a gluten-containing diet and re-inducing the disease symptoms.

In one embodiment, the present invention provides a diagnostic forCeliac Sprue that is a blood test. As discussed above, the 33-mer canalso be detected in peripheral blood samples, when ingested in smallquantities by Celiac Sprue individuals or at the time of an initialscreening at a physician's office.

In one embodiment, the present invention provides a diagnostic forCeliac Sprue that is based on intestinal biopsy staining. Labeled formsof the 33-mer provided by the present invention (e.g. peptide conjugatedto a fluorescent or other label) can be used to stain intestinal biopsysamples from Celiac Sprue patients. Due to their multivalency andanticipated high affinity for antigen presenting cells and, in turn,inflammatory T cells, such peptides can be used to detect the presenceof disease specific immune cells in biopsy tissue. Of particularrelevance is the use of such assays to identify patients whose diseaseis in remission as a result of a gluten-free diet. As noted above,current clinical practices are unable to diagnose a patient when he orshe is on a gluten-free diet, and require that the patient be subjectedto the discomfort of a gluten containing diet for a significant timeperiod.

In one embodiment, the present invention provides a diagnostic forCeliac Sprue in which labeled forms of the 33-mer are used to detectdisease specific immune cells in peripheral blood.

In one embodiment, the present invention provides a diagnostic forCeliac Sprue that is based on an oral mucosa challenge. Inflammatorypeptides from gluten can be used to detect Celiac Sprue by localchallenge on oral mucosa of patients (see Lahteenoja et al., 2000, Am.J. Gastroenterol. 95: 2880). Given the proteolytic resistance andimmunogenicity of the 33-mer, the 33-mer can be especially useful in adiagnostic procedure in which the peptide is contacted with the oralmucosa of an individual, and a diagnosis of Celiac Sprue is made ifinflammation results. Again, a particular advantage of such a test wouldbe its sensitivity to detect a patient whose disease is in remission dueto a gluten-free diet.

In one embodiment, the diagnosis involves detecting the presence of Tcells reactive with the 33-mer or a deamidated counterpart thereof, or atTGase-linked counterpart thereof in a tissue, bodily fluid, or stool ofan individual. T cells can also be detected by proliferation in responseto exposure to an antigen provided by the present invention andpresented by autologous or suitable allogeneic antigen presenting cells.The presence of such reactive T cells indicates the presence of anon-going immune response. The antigen used in the assays may be thecomplete 33-mer, deamidated counterpart, or a tTGase-linked counterpart;or peptides derived therefrom, usually such peptides will be at leastabout 12 amino acids in length. A subset of peptides may be prepared, ora mixture that encompasses the complete sequence. Overlapping peptidesmay be generated, where each peptide is frameshifted from 1 to 5 aminoacids, thereby generating a set of epitopes.

Quantitation of T cells can be performed by determining cognate bindingof the T cell receptor present on a cell, to an MHC/peptide complex,e.g. using Class I or Class II MHC tetramers (see Altman et al. Science(1996) 274: 94-96; McMichael and O'Callaghan J Exp Med. (1998) 187:1367-1371). MHC Tetramers are complexes of the soluble fragments of fourMHC molecules, which are associated with a specific peptide. Thetetramer may be bound to a fluorochromes or other detectable label. (seeOgg et al. (1998) Curr Opin Immunol. 10: 393-396). The tetramer maycomprise a soluble fragment of HLA-DQ2 [DQ(a1*0501, b1*02)] and/or DQ8[DQ(a1*0301, b1*0302)] molecule, or other MHC types appropriate for theindividual being tested.

The diagnosis may determine the level of reactivity, e.g. based on thenumber of reactive T cells found in a sample, as compared to a negativecontrol from a naive host, or standardized to a data curve obtained fromone or more positive controls. In addition to detecting the qualitativeand quantitative presence of antigen reactive T cells, the T cells maybe typed as to the expression of cytokines known to increase or suppressinflammatory responses. While not necessary for diagnostic purposes, itmay also be desirable to type the epitopic specificity of the reactive Tcells, particularly for use in therapeutic administration of peptides.

In another embodiment, the diagnosis involves detecting the presence ofan antibody, reactive with the 33-mer or a deamidated counterpartthereof, or a tTGase-linked counterpart thereof in a tissue, bodilyfluid, or stool of an individual. An antibody is detected by, forexample, an agglutination assay using an antigen provided by the presentinvention. Samples may be obtained from patient tissue, which may be amucosal tissue, including but not limited to oral, nasal, lung, andintestinal mucosal tissue, a bodily fluid, e.g. blood, sputum, urine,phlegm, lymph, and tears. Also included in the term are derivatives andfractions of such fluids. Blood samples and derivatives thereof are ofparticular interest. One advantage of the present invention is that theantigens provided are such potent antigens that the diagnostic methodsof the invention can be employed with samples (tissue, bodily fluid, orstool) in which a Celiac Sprue diagnostic antibody, peptide, or T cellis present in very low abundance. This allows the methods of theinvention to be practiced in ways that are much less invasive, much lessexpensive, and much less harmful to the Celiac Sprue individual.

Measuring the concentration of specific antibodies in a sample orfraction thereof may be accomplished by a variety of specific assays, asare known in the art. In general, the assay will measure the reactivitybetween a patient sample, usually blood derived, generally in the formof plasma or serum. The patient sample may be used directly, or dilutedas appropriate, usually about 1:10 and usually not more than about1:10,000. Immunoassays may be performed in any physiological buffer,e.g. PBS, normal saline, HBSS, dPBS, etc.

In one embodiment, a conventional sandwich type assay is used. Asandwich assay is performed by first attaching the peptide to aninsoluble surface or support. The peptide may be bound to the surface byany convenient means, depending upon the nature of the surface, eitherdirectly or through specific antibodies. The particular manner ofbinding is not crucial so long as it is compatible with the reagents andoverall methods of the invention. They may be bound to the platescovalently or non-covalently, preferably non-covalently.

In some cases, a competitive assay will be used. In addition to thepatient sample, a competitor to the antibodies is added to the reactionmix. The competitor and the antibodies compete for binding to theantigenic peptide. Usually, the competitor molecule will be labeled anddetected as previously described, where the amount of competitor bindingwill be proportional to the amount of antibodies present. Theconcentration of competitor molecule will be from about 10 times themaximum anticipated antibodies concentration to about equalconcentration in order to make the most sensitive and linear range ofdetection.

An alternative protocol is to provide anti-patient antibodies bound tothe insoluble surface. After adding the sample and washing awaynon-specifically bound proteins, one or a combination of the testantigens are added, where the antigens are labeled, so as not tointerfere with binding to the antibodies. Conveniently, fused proteinsmay be employed, where the peptide sequence is fused to an enzymesequence, e.g.—galactosidase.

The subject methods are useful not only for diagnosing Celiac Sprueindividuals but also for determining the efficacy of prophylactic ortherapeutic methods for Celiac Sprue as well as the efficacy of foodpreparation or treatment methods aimed at removing glutens or similarsubstances from food sources. Thus, a Celiac Sprue individualefficaciously treated with a prophylactic or therapeutic drug or othertherapy for Celiac Sprue tests more like a non-Celiac Sprue individualwith the methods of the invention. Likewise, the antibodies or T cellresponders, e.g. T cell lines, of the invention that detect the toxicgluten oligopeptides of the invention are useful in detecting gluten andgluten-like substances in food and so can be used to determine whether afood treated to remove such substances has been efficaciously treated.

These and other diagnostic methods of the invention can be practicedusing the novel peptides and antibodies provided by the invention.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication, patent, or patent application were specifically andindividually indicated to be incorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the inventor to comprise preferredmodes for the practice of the invention. It will be appreciated by thoseof skill in the art that, in light of the present disclosure, numerousmodifications and changes can be made in the particular embodimentsexemplified without departing from the intended scope of the invention.Moreover, due to biological functional equivalency considerations,changes can be made in methods, structures, and compounds withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

What is claimed is:
 1. A method for treating a foodstuff comprisingwheat gluten proteins, said method comprising contacting said foodstuffcomprising wheat gluten proteins in vitro with a glutenase, wherein saidglutenase has a k_(cat)/K_(m) of at least 250 s⁻¹/m⁻¹ for cleaving oneor both of the oligopeptides of SEQ ID NO:3 and SEQ ID NO:12 underphysiologic conditions into fragments shorter than 8 amino acids; toprepare a pre-treated foodstuff that is less toxic to a Celiac sprueand/or dermatitis herpetiformis patient, relative to the untreatedfoodstuff, and contains enzymatically active glutenase when ingested bysaid patient.
 2. The method of claim 1, wherein the glutenase is apeptidase selected from the group consisting of a prolyl endopeptidase(PEP); a PEP homolog; an endoproteinase from a developing grain of agluten-containing cereal; a brush border enzyme; a dipeptidylcarboxypeptidase; and a dipeptidyl peptidase
 4. 3. The method of claim2, wherein the peptidase is selected from the group consisting of a PEPfrom Flavobacterium meningosepticum, Aeromonas hydrophila, Sphingomonascapsulata, and Lactobacilli; a dipeptidyl carboxypeptidase fromPseudomonas, Streptomyces, and Aspergilli; a dipeptidyl peptidase IVfrom Prevotella albensis, Porphyromonas gingivalis, Lactobacillushelveticus, and Lactococcus; a cysteine proteinase B from Hordeumvulgare; a PEP homolog from Aeromonas punctata, Novosphingobiumcapsulatum, Pyrococcus furiosus, E. coli, and Myxococcus xanthus; and anendoproteinase from a developing grain of wheat, barley, and rye.
 4. Themethod according to claim 1, wherein said glutenase is formulated with apharmaceutically acceptable excipient.
 5. The method according to claim1, wherein said glutenase is stable to acid conditions in the stomach ofsaid patient.
 6. The method according to claim 1, wherein said glutenaseis contained in a formulation that comprises an enteric coating.
 7. Themethod of claim 1, wherein the glutenase is formulated in a solidpreparation.
 8. A method for treating a gluten-containing foodstuff torender said foodstuff less toxic to a Celiac Sprue and/or dermatitisherpetiformis patient, said method comprising: contacting said foodstuffin vitro with a glutenase, wherein said glutenase has a k_(cat)/K_(m) ofat least 250 s⁻¹/m⁻¹ for cleaving one or both of the oligopeptides ofSEQ ID NO:3 and SEC) ID NO:12 under physiologic conditions intofragments shorter than 8 amino acids; to form a pre-treated foodstuffthat is then ingested by said patient, wherein said pre-treatedfoodstuff contains enzymatically active glutenase during ingestion, andwherein immunogenic gluten peptides in gluten-containing foodstuffs arecleaved into non-toxic fragments.
 9. The method of claim 8, wherein saidglutenase is a peptidase selected from the group consisting of a prolylendopeptidase (PEP); a PEP homolog; an endoproteinase from a developinggrain of a gluten-containing cereal; a brush border enzyme; a dipeptidylcarboxypeptidase; and a dipeptidyl peptidase
 4. 10. The method of claim9, wherein said peptidase is selected from the group consisting of a PEPfrom Flavobacterium meningosepticum, Aeromonas hydrophila, Sphingomonascapsulata, and Lactobacilli; a dipeptidyl carboxypeptidase fromPseudomonas, Streptomyces, and Aspergilli; a dipeptidyl peptidase IVfrom Prevotella albensis, Porphyromonas gingivalis, Lactobacillushelveticus, and Lactococcus; a cysteine proteinase B from Hordeumvulgare; a PEP homolog from Aeromonas punctata, Novosphingobiumcapsulatum, Pyrococcus furiosus, E. coli, and Myxococcus xanthus; and anendoproteinase from a developing grain of wheat, barley, and rye. 11.The method according to claim 8, wherein said glutenase is formulatedwith a pharmaceutically acceptable excipient.
 12. The method accordingto claim 8, wherein said glutenase is stable to acid conditions in thestomach of said patient.
 13. The method according to claim 8, whereinsaid glutenase is contained in a formulation that comprises an entericcoating.
 14. The method of claim 8, wherein the gluten-containingfoodstuff comprises wheat gluten proteins.
 15. The method of claim 8,wherein the glutenase is formulated in a solid preparation.
 16. A methodfor treating a foodstuff comprising wheat gluten protein to render saidfoodstuff less toxic to a Celiac Sprue and/or dermatitis herpetiformispatient, said method comprising: contacting said foodstuff comprisingwheat gluten protein in vitro with a prolyl endopeptidase, wherein saidprolyl endopeptidase has a k_(cat)/K_(m) of at least 250 s⁻¹/m⁻¹ forcleaving one or both of the oligopeptides of SEQ ID NO:3 and SEQ IDNO:12 under physiologic conditions into fragments shorter than 8 aminoacids; to form a pre-treated foodstuff that contains enzymaticallyactive prolyl endopeptidase; and administering said pre-treatedfoodstuff to said patient, wherein immunogenic gluten peptides in thewheat gluten proteins are cleaved into non-toxic fragments.
 17. Themethod of claim 16, wherein the patient has celiac sprue and exhibitsone or more symptoms from the group consisting of fatigue, chronicdiarrhea, malabsorption of nutrients, weight loss, abdominal distension,and anemia.
 18. The method of claim 16, wherein the patient hasdermatitis herpetiformis that manifests as clusters of intenselypruritic vesicles, papules, and urticaria-like lesions on said patient'sskin.
 19. The method of claim 16, wherein the glutenase is formulated ina solid preparation.